OXIDATION OF MALATE BY ISOLATED
PLANT MITOCHONDRIA
by
NAJAT ALI AL-SANE B.Sc., M.Sc,
January 1981
A thesis submitted for the degree of
Doctor of Philosophy of the University of London
and for the Diploma of Imperial College
Department of Botany and Plant Technology, Imperial College London SW7. -2-
ABSTRACT
The characteristics of malate oxidation by Jerusalem artichoke mitochondria were studied with special attention to the influence of added coenzymes. Thiamine pyrophosphate was found to increase the rate of oxygen uptake suggesting that one of the factors regulating the rate of malate oxidation was the conversion of pyruvate, which is produced as a result of malic enzyme activity, to acetyl-CoA which could subsequently remove oxaloacetate produced as a result of malate dehydrogenase activity thus displacing the equilibrium of the malate + dehydrogenase reaction. Exogenous NAD stimulated oxygen uptake in the presence of malate and had less effect when citrate was the sub- strate.
Piericidin A severely inhibited the oxidation of malate in the presence of oxaloacetate. These results suggest that under such con- ditions only the piericidin A-sensitive pathway was involved in the oxidation of NADH produced from malate.
The effect of n-butyl malonate on the rate of malate oxidation was also studied and results obtained showed that malate oxidation was sensitive to this inhibitor, in the absence of NAD+, while the
NAD+-stimulated rate was not affected by n-butylmalonate. From these results it was concluded that malate oxidation by Jerusalem artichoke mitochondria, takes place through two pathways, one located in the matrix space (transport-dependent) and is associated with the internal
NADH dehydrogenase system. And the other takes place in the outer compartment (transport-independent) and depends on added NAD . Oxi- dation through this pathway is associated with the external NADH de- hydrogenase system. -3-
Endogenous pyridine nucleotide contents were determined for washed and purified Jerusalem artichoke and mung bean mitochondria.
Mung bean mitochondria were found to contain twice that of Jerusalem artichoke mitochondria. Thiamine pyrophosphate contents were also determined for both types of mitochondria.
Further purification of mitochondria was carried out using
Percoll density gradient. Malate oxidation by purified Jerusalem artichoke mitochondria was stimulated by added NAD+. In addition
Jerusalem artichoke mitochondria could reduce exogenous NAD+ when malate was the substrate, but not when citrate was the substrate.
The enzymes responsible for malate oxidation were purified from isolated Jerusalem artichoke mitochondria and some of their kinetic properties were studied. Purified malic enzyme showed a requirement 2+2+ + for Mn or Mg and required NAD as a cofactor. Malate dehydrogen- ase was found to be present in a large quantity when compared with + + malic enzyme and required NAD as a cofactor. The IC^ for NAD of both enzymes were almost of the same magnitude. -4-
ACKNOWLEDGEMENT
First of all I should like to express my sincere gratitude to
Dr.J.M. Palmer for his great help and patient encouragement through- out this investigation.
My thanks are due to Miss Suzanne Cheston for typing the manu- script and to Mrs. Jill Farmer for her help during this study.
I am in debt to my family for their financial and moral support.
I would like to extend my thanks to the University of Kuwait for the financial support. -5-
CONTENTS
ABSTRACT 2
ACKNOWLEDGEMENTS 4
ABBREVIATIONS 8
SYMBOLS 9
LIST OF FIGURES 10
LIST OF TABLES 12
INTRODUCTION
1. General Characteristics of Plant Mitochondria 13
a. Cyanide-resistant pathway 13
b. NADH-dehydrogenase Systems 16
c. Malate oxidation 18
2. Location of enzymes responsible for Malate Oxidation ... 19
a. Dual location 20
b. Transmembrane transhydrogenase 22
3. Purification of Mitochondria 24
4. Aim of the Study 25
MATERIALS AND METHODS
1. Chemicals 27
2. Media Used 27
3. Isolation of Mitochondria 28
a. Jerusalem artichoke mitochondria 28
b. Potato mitochondria 29
c. Mung bean mitochondria 29
d. Rat liver mitochondria 30
4. Assays and analytical procedures 30
a. Oxygen consumption 30 -6-
b. Determination of ADP/O and respiratory control
ratios 31
c. Determination of ADP and AMP concentration 31
d. Determination of protein contents 32
e. Activation of succinate dehydrogenase 32
f. Succinate dehydrogenase assay 32
g. Succinate cytochrome c reductase assay 33
h. Reduction of exogenous NAD+ 33
5. Purification of Mitochondria 34
6. Determination of endogenous NAD+ contents 35
7. Determination of endogenous TPP contents 36 + 8. Purification of NAD -linked malic enzyme 36
a. Isolation of mitochondria 36
b. Sonication 37
c. Ammonium Sulphate fractionation 37
d. Gel filtration 37
e. DEAE-Sephadex 38
f. Assay of enzyme activity 38
9. Purification of NAD+-linked malate dehydrogenase 39
a. Ammonium sulphate fractionation 39
b. Sephadex S-200 40
c. Sephadex G-25 40
d. DEAE-Sephadex 40
e. Assay of malate dehydrogenase 41
RESULTS
1. General characteristics of Jerusalem artichoke
mitochondria 42
2. Pyruvate oxidation 46
3. Malate oxidation 48 -7-
a. Effect of thiamine pyrophosphate 50 + b. Effect of exogenous NAD 53
c. Effect of piericidin A 56
d. Effect of butyl malonate 58
e. Effect of added oxaloacetate 64
4. Purification of Mitochondria 68
a. Integrity of mitochondrial preparations 73
b. Malate oxidation 80
c. Endogenous NAD+ and TPP contents 83
d. Permeability to NAD+ 85 + e. Reduction of exogenous NAD 87
5. Purification of enzymes responsible for malate
oxidation 91
a. Purification of NAD+-malic enzyme 91
b. Purification of NAD+-malate dehydrogenase V.. 99
DISCUSSION 108
1. Effect of Inhibitors 110
a. Effect of piericidin A 110
b. Effect of n-butyl malonate 112
c. Effect of oxaloacetate 113
2. Effect of exogenous NAD+ 115
3. Reduction of exogenous NAD+ 117
4. Purification of mitochondria 119
5. Malate oxidizing enzymes 121
REFERENCES 125
APPENDIX Palmer, J.M., Cowley, R.C. & Al-Sane, N.A. (1978). The
inhibition of malate oxidation by oxaloacetate in
Jerusalem artichoke mitochondria. 'Plant Mitochondria'
(Ducet, G. & Lance, C. eds.) Elsevier, Amsterdam. -8-
ABBREVIATIONS
ADP Adenosine-5'-diphosphate
AMP Adenosine-5'-monophosphate
ATP Ad eno s ine-5'-tr ipho sphat e
BM n-butylmalonate
BSA Bovine serum albumin (fraction V)
DCIP 2,6-Dichlorophenol-indophenol
DTT Dithiothretol
EDTA Ethylenediamine tetraacetic acid
MOPS 3-(N-morpholino) propane sulphonic acid
NAD+ Nicotinamide-adenine dinucleotide (oxidized)
NADH Nicotinamide-adenine dinucleotide (reduced)
OAA Oxaloacetate
P/A Piericidin A
PEP Phosphoenol pyruvate
PK Pyruvate kinase
PMS Phenazine methosulphate
S.E. Standard error
TES N-tris(hydroxymethyl)-methyl-2-aminoethane sulphonic acid
TPP Thiamine pyrophosphate
Triton X 100 Octylphenoxypolyethoxyethanol SYMBOLS
maximum velocity of reaction
Michaelis constant, the concentration of substrate permitting half V ° max
-2 acceleration due to gravity (981 cm.s ) -10-
LIST OF FIGURES
page
1 Malate oxidation by Jerusalem artichoke and rat liver mitochondria 45
2 Pyruvate oxidation by Jerusalem artichoke mito- chondria 47
3 Malate oxidation by Jerusalem artichoke mito- 49 chondria
4 Effect of cofactors on malate oxidation 51 + 5 Effect of exogenous NAD and TPP on malate oxi- dation 52 6 Effect of piericidin A on malate oxidation 57
7 Effect of n-butylmalonate on malate oxidation 59
8 Effect of n-butylmalonate on NAD+-stimulated
rate 62
9 Effect of oxaloacetate on citrate oxidation 66
10 Effect of oxaloacetate on malate oxidation 67
11 Development of Percoll density gradient 71
12 Distribution of Jerusalem artichoke mitochondria on 20% Percoll density gradient 72 13 Distribution of Jerusalem artichoke mitochondria on 18% Percoll density gradient 74
14 Distribution of mung bean mitochondria on Percoll density gradient 75
15 Distribution of potato mitochondria on Percoll density gradient 76
16 Exogenous NAD+ reduction by Jerusalem artichoke mitochondria 88
17 Purification of malic enzyme 93
18 Activity of malic enzyme as a function of NAD+ concentration in the presence of 10 mM Malate 95
19 Activity of malic enzyme as a function of NAD+ concentration in the presence of 50 mM Malate 96 -11-
page
20 Linweaver-Burk plot of reaction of velocity of malic enzyme as a function of NAD concentrations in the presence of MnCl^ 97
21 Linweaver-Burk plot of reaction velocity of malic enzyme as a function of NAD concentrations in the presence of MgCl^ 98
22 The rate of malate dehydrogenase activity as a function of enzyme concentration 102
23 Malate dehydrogenase activity as a function of malate concentration 103
24 Mal^te dehydrogenase activity as a function of NAD concentration 105
25 Linweaver-Burk glot of reaction velocity as a function of NAD concentration 106 26 Linweaver-Burk plot of reaction velocity as a function of malate concentration 107 -12-
LIST OF TABLES
page
1 Rate of oxidation of different substrates 43
2 Stimulation of oxygen uptake by exogenous NAD+ 55
3 Effect of BM on state 3 and NAD+-stimulated rates of malate oxidation 61
4 Effect of BM on enzyme activity 63
5 Cytochrome c reduction by washed and purified mitochondria 77
6 NADH oxidation by washed and purified mito- chondria 79
7 Effect of NAD+ and TPP on malate oxidation by washed and purified Jerusalem artichoke mito- chondria 81
8 Effect of NAD+ and TPP on malate oxidation by washed and purified mung bean mitochondria 82 -f 9 Endogenous NAD and TPP contents 84
10 NAD+ uptake by Jerusalem artichoke mitochondria 86 + 11 Reduction of exogenous NAD by Jerusalem arti- choke mitochondria 89 12 Purification of NAD+-malic enzyme 92
13 Purification of NAD+-malate dehydrogenase 100 INTRODUCTION
1. General Characteristics of Plant Mitochondria
Plant mitochondria were for many years thought to be similar to mammalian mitochondria, where the respiratory chain is arranged in a linear sequence which is shown in the scheme together with the sites of action of inhibitors and sites of ATP synthesis.
Pyruvate ATP ATP ATP ^">51 + t i Isocitrate NADH FMN Fe-S UQ -»• cyt b cyt c cyt aa3 0« /* / + + + t a-ketoglutarate P/A Fe-S antimycin A cyanide rotenone + / FAD
Succinate
In closer examinations, mitochondria isolated from plants and some microorganisms have been shown to have considerably more complex meta- bolic respiratory chain than that found in mammalian mitochondria the major features of which will be discussed.
a. Cyanide-resistant pathway
One of the main differences between mammalian and plant mito- chondria is that electron transport of plant mitochondria is branched and allows for the reduction of molecular oxygen by either of two pathways (Ikuma 1972, Solomos 1977). The term cyanide-resistant res- piration describes the cellular respiration which is insensitive to cytochrome inhibitors such as cyanide which inhibits electron trans- port from cytochrome a^ to oxygen and antimycin A which inhibits electron transport from cytochrome b to cytochrome c. Mitochondria isolated from resistant tissues contain both the cyanide sensitive -14-
electron transport system which is coupled to phosphorylation, and
the cyanide-insensitive pathway which is not coupled to ATP synthesis.
Bendall and Bonner (1971) observed that metal complexing agents
such as thiocyanide, 8-hydroxyquinoline and a-a-dipyridyl inhibit the
cyanide resistant pathway in skunk cabbage mitochondria, and it was
suggested that the alternative pathway comprises a non-heme iron pro-
tein. Bahr and Bonner (1973a) showed that these compounds inhibit the
main respiratory pathway as well.
Schonbaum et al. (1971) reported that the cyanide- and antimycin
A-insensitive alternate pathway is specifically inhibited by various
hydroxamic acids (R-CONHOH), and that the most effective are m-chlor-
obenzhydroxamic acid and m-iodobenzhydroxamic acid. They also re-
ported that benzhydroxamic acids have no effect on mitochondria lack-
ing the cyanide-resistant pathway such as potato mitochondria.
The ability of hydroxamic acids to chelate transition metals was
considered to be the most likely mechanism for their inhibitory eff-
ect. Schonbaum et al. (1971) found that when cyanide-insensitive
mitochondria were treated with m-CLAM then washed with 0.3 M mannitol,
the cyanide resistant respiration was restored. A further support
for this observation was reported by Henry et al. (1973) who showed 3+ that the addition of Fe counteracted the effect of hydroximate.
Siedow and Girvin (1980) showed that propylgallate specifically
inhibited the alternative pathway of electron transfer in isolated mung bean mitochondria. And they suggested that it acts at or very
near the site of action of hydroxamic acid (SHAM) as it has the abil-
ity to chelate ferric ions.
The degree of resistance to cyanide varies with the species. For
example, skunk cabbage mitochondria are highly cyanide-resistant,
mung bean mitochondria are partly cyanide-resistant, while mito- -15-
chondria isolated from white potato tubers are highly cyanide-sensi- tive (Bendall and Bonner 1971; Bahr and Bonner 1973a,b).
Although much attention has been focused on the respiratory chain of higher plants and in particular upon the fact that many species possess a pathway of electron transport to oxygen which is not via cytochrome oxidase and which is insensitive to cyanide, the exact nature of this alternate oxidase and the precise point of interaction has not been established.
The alternate pathway is generally held to branch from the main respiratory chain at the ubiquinone region (Solomos 1977). Storey
(1976) carried out oxygen pulse experiments using skunk cabbage mito- chondria and potato mitochondria, in the presence of saturating con- centrations of CO and reported that the rate of ubiquinone oxidation was similar in both cases which suggested that ubiquinone is common to both oxidases. Storey (1976) also reported that the mid potential flavoprotein (Fpma) may be the first component of the alternate path- way.
Huq and Palmer (1978) observed that in Arum mitochondria, the oxidation of exogenous NADH was completely insensitive to cyanide, which shows that electrons from exogenous NADH and from endogenous
NADH appear to have equal access to the cyanide resistant oxidase.
On the other hand when using mitochondria from cassava or sweet pot- ato, they observed that external NADH was unable to supply electrons to the alternative oxidase, while succinate Could be oxidized by the alternative oxidase. They stated that if ubiquinone serves as a common pool which receive electrons from different dehydrogenases and distributes them to the different oxidases, it is difficult to explain how electrons flowing from exogenous NADH would be excluded from the -16-
alternative oxidase. Thus suggesting the presence of two functional pools of ubiquinone.
b. NADH dehydrogenase systems
One of the major differences between plant and mammalian mito- chondria is that mammalian mitochondria cannot oxidize exogenously added NADH (Seifart andBenecke 1975). And studies showed that NADH does not traverse the inner mitochondrial membrane and is unable to
supply electrons to respiratory chain (Greenspan and Purvis 1965;
Klingenberg and Pfaff 1966). The ability of plant mitochondria to oxidize exogenous NADH is attributed to a dehydrogenase located on
the outer surface of the inner membrane, and is connected to the elec-
tron transport chain. This pathway is inhibited by antimycin A, but is insensitive to site I inhibitors such as rotenone, and bypasses
the first site of phosphorylation (Coleman and Palmer 1972; Douce et al. 1973; Day and Wiskich 1974a).
Plant mitochondria appear to have two external NADH dehydrogen- ase systems, one is located on the outer surface of the inner membrane and the other is associated with the outer membrane consisting of
flavoprotein and cytochrome (Moreau and Lance 1972; Douce et al.
1973). Reducing equivalents can be transported from the dehydrogen-
ase on the outer membrane to the respiratory chain only in the pres- ence of soluble cytochrome c. This pathway is insensitive to inhib-
ition by rotenone or antimycin A (Douce et al. 1973; Palmer and
Coleman 1974).
The NADH dehydrogenase system responsible for the oxidation of endogenous NADH in plant mitochondria appear to be more complex than
that of mammalian mitochondria.
In mammalian mitochondria, the NADH produced in the matrix as a -17-
+ result of NAD -linked reactions is oxidized via rotenone-sensitive
NADH dehydrogenase. This dehydrogenase is a part of the respiratory chain and electron flow along this chain is coupled to three sites of
ATP synthesis. While in mammalian mitochondria the oxidation of en- dogenous NADH is completely inhibited by a low concentration of roten- one or piericidin A? plant mitochondria needed higher concentrations to cause inhibition, and inhibition caused is only partial (Wilson and
Hanson 1969; Brunton and Palmer 1973; Day and Wiskich 1974a,b;
Pomeroy 1974; Palmer and Arron 1976).
The variability in response to rotenone or piericidin A and the decrease in ADP:0 ratio observed in association with oxidation of
NAD+-linked substrated lead to the conclusion that endogenous NADH is oxidized via two pathways. One is sensitive to rotenone and coupled to three sites of ATP synthesis and the other is insensitive to rot- enone and coupled to two sites of ATP synthesis (Palmer and Coleman
1974).
Brunton and Palmer (1972) using wheat mitochondria, and extern- ally added oxaloacetate to inhibit malate oxidation, concluded that there are two kinetically distinct compartments in the matrix, one containing malic enzyme and the rotenone sensitive NADH dehydrogenase "ing + and the other contain^the rest of NAD -linked enzymes including mal- ate dehydrogenase and the rotenone-insensitive NADH dehydrogenase.
Palmer and Arron (1976) using Jerusalem artichoke mitochondria and malate as a substrate, reported that there are two pathways for internal NADH oxidation. They also observed that the oxidation of malate was more sensitive to inhibition by piericidin A than that of other NADH+-linked substrates. They also provided evidence suggest- + ing that endogenous NAD reduced by either citrate or 2-oxoglutarate can be oxidized by piericidin A-resistant NADH dehydrogenase, while -18-
the NADH produced from malate oxidiation did not have immediate access
to piericidin A-resistant pathway unless the oxaloacetate, produced as a result of malate dehydrogenase activity is removed, suggesting the presence of two compartments in the matrix as was shown for wheat raotochondria (Brunton and Palmer 1972).
Sotthibandhu and Palmer Q975) investigating the oxidation of
NADH"-linked substrates In the presence of weak acid uncouplers, ob- served that the addition of AMP brought about a simultaneous oxidation of endogenous pyridine nucleotide and reduction of cytochrome b. They concluded from their results that AMP stimulated electron flow from
NADH to cytochrome b via a piericidin A-sensitive pathway and because the stimulation was not inhibited by bongkrekic acid they concluded that AMP does not have to enter tha mitochondria to cause the stimul- ation.
It seems that the level of AMP in the cell would activate the phosphorylating internal NADH and thus Increase the supply of ATP
(Palmer and Coleman 1974; Palmer 1979).
c. Malate oxidation
Malate oxidation in plant mitochondria is a much more complex process than that in mammalian mitochondria. In mammalian mitochon- dria, malate oxidation is achieved by a single enzyme, the malate de- hydrogenase and the continual oxidation of malate is dependent on the , removal of oxaloacetate produced. As the reaction equilibrium (Keq -12
2.3 x 10 ) favours malate formation from oxaloacetate. The removal of oxaloacetate can be achieved either by condensation with acetyl-
CoA or by transamination with glutamate. Plant mitochondria can oxi- dize malate without the necessity for a system to remove oxaloacetate f produced (Lance et al. 1967; Wiskich and Bonner 1965), which suggest that \ -19-
plant mitochondria either have a system for removing oxaloacetate or the presence of an additional enzyme capable of oxidizing malate.
Macrae and Moorhouse (1970) and Macrae (1971a,b) discovered the presence of malic enzyme in plant mitochondria which not only oxidizes malate but gives pyruvate as product. Oxidation of malate via malic enzyme gives rise to pyruvate which is decarboxylated by pyruvate dehydro- genase to give acetyl-CoA which in turn will condense with oxaloace- tate to give citrate (Macrae 1971b; Brunton and Palmer 1973; Lance et al. 1967).
2. Location of enzymes responsible for malate oxidation
Many workers have observed that exogenously added NAD+ stimu- lated the rate of malate oxidation, and that piericidin A (or roten- one) only caused a partial inhibition which is relieved upon addition of ImM NAD+ (Coleman and Palmer 1972; Brunton and Palmer 1973; Day and Wiskich 1974a,b; Palmer and Arron 1976; Day and Wiskich 1978;
Neuberger and Douce 1978).
Although it is generally agreed that plant mitochondria contain
two enzymes which are responsible for the oxidation of malate: namely, malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) and + + NAD -linked malic enzyme (L-malate: NAD oxidoreductase (decarboxy-
lating), EC 1.1.1.39)? the precise relationship they have with the + NADH dehydrogenase and location of the NAD -linked malic enzyme are matters of controversity.
Two interpretations for the response of malate oxidation to
added NAD+ wereoffered. Palmer and colleagues have suggested that
NAD+-linked malic enzyme located in the intermembrane compartment + reduced externally added NAD which is then reoxidized by a specific
NADH dehydrogenase located near the external face of the inner mem- -20-
brane and is linked to the respiratory chain. In contrast, Day and
Wiskich (1974a,b) and Day and Wiskich (1978) indicated that malate needed to penetrate through the inner membrane to be oxidized and they proposed that a transmembrane transhydrogenase was responsible for the + reduction of exogenous NAD and the resultant NADH can be oxidized by the external NADH dehydrogenase.
More recently, Neuberger and Douce (1978), reported that the state 3 rate of malate oxidation was stimulated by the addition of exogenous NAD and that ADP/0 ratio was not affectedo By measuring the uptake of labelled NAD they suggested that exogenously added
NAD+ was taken up by plant mitochondria and that the rate of uptake is affected by the initial concentration of NAD+ present in the mito- chondrial matrix. They suggested that the for NAD+ to be 0.3mM.
a. Dual location of malic enzyme
The oxidation of malate takes place in the matrix of the mito- chondria by either malate dehydrogenase or malic enzyme; and under certain conditions malate oxidation can take place in the inter- membrane space by part of the malic enzyme located in the intermem- brane compartment (Coleman and Palmer 1972; Brunton and Palmer 1973;
Palmer and Arron 1976).
The suggestion of Palmer and colleagues that some of the NAD+- linked malic enzyme is located in the intermembrane compartment was based on the following observations.
1. In the absence of exogenous NAD+ a great proportion of the products of malate oxidation was aspartate (in the presence of gluta- mate); while in the presence of NAD+ and piericidin A equal amounts of aspartate and pyruvate were produced (Coleman and Palmer 1972;
Palmer and Arron 1976). + If the addition of NAD stimulated malate dehydrogenase as well as malic enzyme, both products would increase in the same proportion.
But under conditions applied pyruvate production was increased. This would suggest that in the presence of piericidin A, added NAD+ would only stimulate malic enzyme.
2. In the presence of piericidin A, the rate of oxidation of + malate was stimulated by added NAD to greater extent than that of other NAD+-linked substrates. Besides phosphate was found to be necessary for the oxidation of malate in the absence of added NAD+, + but not necessary for the stimulation by exogenous NAD (Palmer and
Arron 1976). Thus suggesting that the component of malate oxidizing system stimulated by exogenous NAD+ was independent of malate trans- port through the inner membrane.
3. In the absence of added NAD+, malate oxidation which was sensitive to piericidin A was also inhibited by n-butylmalonate (an inhibitor of substrate transport; (Phill ips and Williams 1973); while in the presence of exogenous NAD+ malate oxidation was almost un- affected (Coleman and Palmer 1972).
4. Using ferricyanide as an electron acceptor, Coleman and
Palmer (1972) obtained results which indicate that in the presence of rotenone or antimycin A, malate could reduce ferricyanide by a mech- anism which is dependent on the addition of NAD+.
Palmer and Arron (1976) reported that the addition of NAD+ be- fore antimycin A resulted in a greater stimulation of the ferricyanide + 1 reduction than if NAD was added after antimycin A. They suggested + that the addition of NAD activated oxidation mediated by internally located NADH dehydrogenase. -22-
b. Transmembrane transhydroganase
Day and Wiskich (1974a,b) and Day and Wiskich (.1978) suggested that malate oxidation takes place in the matrix and a transmembrane + hydrogen transfer between internal NADH and external NAD was involved.
They based their view on the following observations: + 1. Malate oxidation both in the presence and absence of NAD and rotenone was equally sensitive to n-butylmalonate and equally dependent on added phosphate Can activator of substrate translocation).
Observations that are different from those obtained by Coleman and
Palmer (1972).
Evidence in the literature suggest that butylmalonate can dir- ectly inhibit malate dehydrogenase over the same concentration range employed to inhibit malate translocation into the mitochondria
(Phillips and Williams 1973). Thus Inhibition of NAD+ induced stimu- lation of malate oxidation might be due either to the inhibition of malate translocation or to the direct Inhibition of the enzyme re- sponsible for the oxidation of malate. In addition phosphate stimu- lation might be due to the known ability of phosphate to activate the mitochondrial malate dehydrogenase (Blonde et al. 1967).
2. Upon addition of NAD+, rotenone inhibition of malate oxi- dation was almost completely relieved and ADP/O ratios were lowered.
In addition, in the presence of cytochrome c, the addition of NAD+ relieved antimycin A inhibition. Under these conditions malate oxi- dation becomes linked to external NADH oxidation.
3. Addition of NAD+ relieved rotenone inhibition when citrate or a-ketoglutarate were used as a substrate, in a similar manner as to that of malate. They argued that If the response of malate oxidation to added NAD+ is due to the localization of the malic enzyme in the intermembrane compartment, then the response of other NAD+-linked -23-
dehydrogenases to the addition of NAD in the same manner as that of malate oxidation would suggest that all NAD+-linked dehydrogenases are located in the intermembrane compartment, a condition which is un- + acceptable. They concluded that all NAD -linked dehydrogenases in— . + . eluding the NAD -linked malic enzyme are located in the mitochondrial matrix.
Palmer and Arron (1976) carried out a control experiment to which they added piericidin A, then NAD+ (Palmer and Arron 1976, table 2). . • + They observed that the addition of NAD caused stimulation of res- piration when no substrate was added. And when using citrate as a substrate, the stimulation by added NAD+ was not greater than the control. On the other hand, when using malate as a substrate, the addition of NAD+ resulted in a major stimulation of respiration and this rate was above that of the control. This suggests that only malate oxidation was stimulated by added NAD+.
4. Day and Wiskich (1978) studying the kinetic behaviour of + NAD reduction, reported that malate dehydrogenase can reduce exogen- ous NAD+ in intact mitochondria. They observed that when intact mito- chondria were supplied with malate as substrate, they reduce exogen- ous NAD+ rapidly and the rate of reduction declined after one minute then stopped. They attributed the decrease in the rate of reduction to the accumulation of internal NADH. As antimycin A was present in the reaction medium, thus NADH produced is not oxidized via the res- piratory chain. From these results they concluded that a transmem- brane transhydrogenase was involved and they argued that if an exter- nal enzyme was involved, a linear rate of NAD+ reduction would be obtained. At the same time they observed that isocitrate failed to induce external NAD+ reduction in the presence of antimycin A;- -24-
. . + although they observed that addition of NAD relieved rotenone in- hibition of citrate oxidation. They attributed the failure of citrate + .... to reduced exogenous NAD to be due to the inhibition of isocitrate oxidation by the internal NADH level and that isocitrate does not produce enough concentration of NADH to bring about the operation of transmembrane transhydrogenase. On the other hand, Brunton and Palmer
(1972) showed that malate and citrate were oxidized by wheat mito- chondria and produced equal amounts of endogenous NADH both in the presence and absence of KCN. While citrate failed to reduce external
NAD+.
In most of the work carried out, washed mitochondria were used.
When studying the location of enzymes it is essential that the mito- chondria used are pure and intact. 3. Purification of mitochondria
Mitochondria prepared by usual differential centrifugation technique from different sources of higher plants (Bonner 1967) and lower plants (Lambowitz et al. 1972) are to some extent contaminated with other subcellular structures. And in case of mitochondria iso- lated from higher plant leaves are contaminated with chlorophyll
(Douce et al. 1977).
In an attempt to achieve high quality and homogenous mitochondria, lengthy procedures using continuous and discontinuous sucrose gradient centrifugation have been developed (Douce et al. 1972; Day and Hanson
1977c). Although these methods yield intact and pure organelles, they are time consuming and need careful dilution to isosmotic concen- tration of sucrose to avoid physical damage as some mitochondrial pre- parations do not survive the isosmotic dilution (Douce et al. 1972).
Douce et al. (1977) isolated mitochondria from spinach leaves, al- -25-
though their preparation oxidized a variety of substrates with good respiratory control and ADP/O ratios, they were still contaminated with chlorophyll. Day and Hanson (1977c) compared two methods for purification. One was by using sucrose gradient and the other was by centrifuging through a layer of 0.6 M sucrose. While the first one was time consuming the other failed to remove much of the contaminants.
Arron et al. (1979) using linear sucrose gradient, reported that much of the non-mitochondrial protein was removed from the washed prepar- ation, but some degree of respiratory control was lost. This loss was also reported by Day and Hanson (1977c) for the gradient purified mitochondria. They attributed this loss to the time cohsumed in cen- trifugation.
Jackson et al. (1979) described a technique for purifying mito- chondria from green leaves. A three-step discontinuous gradient of
Percoll was used. By this method, they could remove most of the chlorophyll from their washed mitochondria while retaining intactness, respiratory control and ADP/0 ratios.
During this study a technique for purifying mitochondria was de- veloped. This technique consumed less time and proved to be effect- ive. The density medium used was Percoll which is a silica sol having low viscosity, low osmolarity and is non-toxic. Mitochondria purified by this method were found to retain Intactness, respiratory control and
ADP/0 ratios.
4. Aim of the study
The aim of this study was to investigate the relationship between + . . the NAD -linked oxidation of malate and the operation of the internal facing and external facing NADH dehydrogenase systems. It has been
shown that in plant mitochondria there are two pathways for malate -26-
oxidation, by mitochondrial malic enzyme via the piericidin A insen- sitive pathway and by malate dehydrogenase via the piericidin A sen- sitive pathway. Use was made of piericidin A and n-butylmalonate to investigate the location of the malic enzyme and malate dehydrogenase within mitochondria isolated from Jerusalem artichoke tubers and to resolve the way which respiration is stimulated by NAD+. -27-
MATERIALS AND METHODS
1. Chemicals
+ NAD , NADH, ADP, and all enzymes used were obtained from
Boehringer Corporation Ltd. (London); 3-(N-morpholino) propane-sul- phonic acid (MOPS) and N-tris (hydroxymethyl-2-aminoethane sulphonic acid (TES) were obtained from Hopkin and Williams; malate, glutamate, antimycin A, Thiamine pyrophosphate (TPP), dithiothreitol (DTT) and bovine serum albumin (fraction V) were from Sigma Ltd. (London); piericidin A was a generous gift from Professor T.P. Singer; n-butyl- malonate (BM) was a generous gift from Professor J. Wiskich. Other substrates and chemicals were obtained from BDH Ltd. (Poole). Sephac- ryl S-200, Sephadex A-25, Sephadex G-25 and Sephadex A-50 and Percoll were from Pharmacia Fine Chemicals (U.K.).
2. Media Used
Isolation medium for Jerusalem artichoke, Potato and mung bean mitochondria
0.5 M sucrose
10 mM MOPS
5 mM EDTA
0.1% (w/v) BSA at pH 7.8
Isolation/wash medium for rat liver mitochondria
0.25 M sucrose
0.1 mM EDTA
10 "mM trls-HCl at pH 7.6
Wash medium for Jerusalem artichoke, Potato and mung bean mito- chondria -28-
0.5 M sucrose
5 mM TES
0.1% (w/v) BSA at pH 7.2
Assay medium for Jerusalem artichoke, Potato and mung bean mito- chondria
0.4 M sucrose
5 mM KH P0, 2o 4 4 mM TES
2.5 mM MgCl2 at pH 7.2
Assay medium for rat liver mitochondria
25 mM sucrose
1 mM EDTA
5 mM TES
10 mM KH P0, 2o 4 0.1 M KC1
10 mM MgCl2 at pH 6.8
The pHfs of the media were adjusted with K0H solution.
3. Isolation of Mitochondria
a. Jerusalem artichoke mitochondria
Jerusalem artichoke (Heli/znbhus tuberosus) tubers were obtained from the Botanical Supply Unit, University of London, cleaned, dried and stored in polyethylene bags at a temperature of 5-9°C until re- quired. Mitochondria were isolated from Jerusalem artichoke tubers the by^method described by Palmer and Kirk (1974), except that a Moulinex mixer 66 (Alenson, France) was used for the disruption of tissue.
300 ml of standard isolation medium were used for 200 g of peeled tubers. 2 mM sodium metabisulphite was included in the isolation -29-
medium to help protect mitochondria from damage by phenolic compounds released during tissue disruption. The homogenate was filtered through two layers of muslin and the filtrate was centrifuged at
48,Q00 g for 3 minutes. The pellet obtained was homogenized gently in a teflon-glass homogenizer using a 100 ml of standard wash medium.
The homogenate was then centrifuged up to 11,000 g and immediately brought to stop, then the supernatant was centrifuged at 48,000 g for
3 minutes. The pellet obtained was suspended in a small amount of wash medium to give 20-30 mg protein per millilitre.
b. Potato mitochondria
Potato tubers CSolarium tuberosum) were purchased from local mar- ket, cleaned, dried and stored at 5-9°C. Procedure for isolation was the same as described for Jerusalem artichoke, except that a low speed centrifugation step (11,000 g for 2 min.) was introduced prior to the first high speed to deposit starch.
c. Mung bean mitochondria
Mung bean seeds (JPhaseulus aureus) were purchased locally and surface sterilized using 1Q% (v/v) sodium hypochlorite for 5 minutes, then washed thoroughly with water. Seeds were sown thinly over a nylon gauze stretched over a circular wire frame suspended in a round plastic bowl containing tap water. The bowls were placed in a dark incubator and the temperature was maintained at 27°C for 4 days.
The hypocotyls were separated from roots and epicotyls by means
of scissors. The hypocotyls were washed several times with, ice-cold
distilled water, to eliminate any contamination. Then cut into sec-
tions of about 2 cm and placed In a pre-cooled porcelain mortar with
isolation medium in a ratio of 1 gram material to 2 ml medium, and
ground by hand with a pestle for 30 seconds. Isolation and wash -30-
media were the same as those used for Jerusalem artichoke except for the metabisulphite which was omitted in this case as it was found to cause damage to mung bean mitochondria. Mitochondria were then iso- lated following the method described for Jerusalem artichoke.
d. Rat liver mitochondria
The method used here was that described by Weinbach (1961). The liver ofafreshly killed Sprague Dowley rat was removed and rinsed in isolation medium. Then cut into strips and homogenized in 50 ml iso- lation medium in a teflon-glass homogenizer. The homogenate was cen- trifuged at 600 g for 10 minutes to remove cell debris. The super- natant was then centrifuged at 12,000 g for 10 minutes. The fluffy material on the surface of the sedimented mitochondria was removed by rinsing the pellet with 1-2 ml of isolation medium. The mitochondria were resuspended in 10 ml of isolation medium and centrifuged at
12,000 g for 10 minutes. The final pellet was resuspended in iso- lation/wash medium to give protein concentration of about 20 mg/ml.
A Sorvall RC-2B centrifuge was utilized for the preparation of mitochondria and all steps were conducted at 0-4°C.
4. Assay and analytical procedures
a. Oxygen consumption
Oxygen uptake was measured using Rank oxygen electrode (Rank
Bros. Bottisham, Cambridge U.K.) connected to a Servoscribe recorder.
Each assay contained 0.8-1.0 mg mitochondrial protein in one ml of standard assay medium. The reaction mixture was stirred by a mag- netic stirrer and temperature was maintained at 25°C by means of cir- culating water-bath. The concentration of oxygen in air-saturated medium was determined for this electrode to be 240 yM (Cowley 1977) and this value was used throughout this study.
b. Determination of ADP/0 and respiratory control ratios
Respiratory control and ADP/0 ratios were calculated from traces obtained using oxygen electrode according to the definition given by
Chance and Williams (1956). ADP/O ratio was calculated as ymole of ADP phosphorylated per yatom of oxygen consumed. The concentration of ADP was determined enzymatically as described below.
c. Determination of ADP and AMP concentrations
The concentrations of ADP and AMP in solution was determined by the method of Joworek et al. CI974) using the Boehringer ADP/AMP test kit. The ADP estimation was based on the enzymatic reaction cata- lyzed by pyruvate kinase (PK) and lactate dehydrogenase (LDH). PK ADP + phosphoenol pyruvate (PEP) * ATP + pyruvate
Pyruvate + NADH + H*; LDH lactate + NAD+
The decrease of NADH, as measured by the change in extinction at
340 nm is proportional to the amount of ADP present. The reaction mixture consisted of 2 ml buffer at pH 7.5 (0.1 M tris-HCl + 0.55 M
K2C03), 0.94 mM PEP, 33.8 mM MgS04, 0.12 M KC1, 0.32 mM NADH, 5 yl of
ADP solution ( approximately 25 mM) and 0.02 ml of LDH (5 mg/ml) . The reactants were mixed well and extinction recorded (Ej).
Then 0.02 ml of PK (2 mg/ml) was added and extinction (E2) was re- corded after the completion of the reaction. The amount of AMP in the
ADP solution was determined by adding the adenylate kinase, which cat- alyzed the reaction
AMP + ATP 2 ADP to the reaction mixture after the concentration of ADP had been de- termined . -32-
Using the extinction coefficient of 6.22 litre, mmole '.cm ' for
NADH at 340 nm, the concentration of ADP (mM) could be calculated.
d. Determination of protein Contents
Protein estimation was carried out using the method of Lowry et al. (1951) after first solubilizing the protein with deoxycholate
(0.4 ml of 10% (w/v) aqueous solution). 5-10 yl of mitochondrial sample containing not more than 200 yg protein was used. 5 ml of 20%
Na2C03 in 0.1 N NaOH was added followed by 0.05 ml of 1% CuSO^ and
0.05 ml of 2% Na/K tartarate. The contents were mixed thoroughly using a vortex mixer and left to stand for 10 minutes. After this period of time 0.5 ml of diluted Folin and Ciocallen's phenol reagent
(1 part phenol reagent + 2 parts water) was added to the test tube while mixing thoroughly, then left to stand for 30 minutes. The ab- sorbance of the coloured solution was read at 600 nm using Unicam SP-
500 spectrophotometer (Pye-Unicam Ltd. Cambridge U.K.). A reference was prepared by using wash medium instead of the sample. Protein con- tent was determined from a calibration curve prepared using crystaline bovine serum albumin as standards, treated in the same way.
e° Activation of succinate dehydrogenase
Two volumes of mitochondria (20 mg/ml) were incubated with 1 vol- ume of 50 mM ATP, 1 volume of 50 mM KH2P0^ (in 0.4 M sucrose at pH
7.2) and MgCl2 to a final concentration of 5 mM; incubation was for
4 minutes at 26°C. Mitochondria were then kept in ice throughout the course of experiment (Cowley 1977).
f. Succinate dehydrogenase assay
Succinate dehydrogenase activity was measured spectrophotometri- cally at room temperature by the phenazinemethosulphate (PMS)-mediated -33-
reduction of DCIP at 600 nm (Bajinsky and Hatefi 1969) utilizing DW-2
spectrophotometer in a split beam mode. The reaction mixture con-
tained 50 mM potassium phosphate (pH 7.4), 0.1% BSA, 0.1 mM EDTA,
70 yM DCIP, 3 mM KCN and 0.013% of Triton X 100. PMS was then added
in a final concentration of 1.65 mM. This was followed by 50 yl of
the sample and the reaction was started by the addition of 20 mM suc-
cinate. Concentrations were in a total volume of 1 ml and succinate
dehydrogenase was activated prior to assaying.
g. Succinate-Cytochrome c reductase
The outer mitochondrial membrane integrity was tested by measur-
ing the cytochrome c reduction. This was measured by following the
increase in absorbance at 550 nm and 540 nm as reference in intact and osmotically disrupted mitochondria (Douce et al. 1972). Mito- chondria were incubated for two minutes in 1 ml assay medium which, contained no sucrose, to assay activity in disrupted mitochondria.
The assay was carried out in 1 ml standard reaction medium Cor reac-
tion medium containing no sucrose) which also contained 3 mM KCN and
0.05 mM cytochrome c. An aliquot of mitochondria containing 20-40 yg protein was used and the reaction was initiated by the addition of
10 mM succinate.
Succinate dehydrogenase was activated by incubating the mito- chondria with 0.2 mM ATP for 5 minutes before assaying. The. extinc-
tion coefficient for cytochrome c of 21.0 litre.mmol *.cm * was used when calculating the activity. Aminco DW-2 dual wavelength spectro- photometer was utilized.
h. Reduction of exogenous NAD*
The reduction of exogenous NAD+ was measured by following the in- crease in absorbance at 340 nm using 374 nm as the reference wave- -34-
length in an Aminco DW-2 dual wavelength spectrophotometer at room temperature, using cuvettes of 1 cm light-path. The reaction was carried out in a 1 ml of standard assay medium containing 0.05 ml + mitochondrial suspension, 500 ng of antimycin A and 1 mM NAD . The reaction was initiated by adding the appropriate substrate. The ex- tinction coefficient of 3.73 litre.mmol ^.cm * was used to calculate the change in NADH concentration.
5. Purification of mitochondria
The plant material used in these experiments included Jerusalem artichoke tubers, potato tubers and mung bean hypocotyls. Mitochon- dria were prepared by methods that have been described before and the washed mitochondria were suspended in a small amount of the standard wash medium.
Purification of mitochondria was carried out by using a Percoll gradient, this was attained by preparing a Percoll mixture of 20% or
18% (v/v) of Percoll (of that supplied by Pharmacia) in 0.5 M sucrose and 5 mM TES (pH 7.2). Percoll mixture (40 ml) was placed in a 50 ml centrifuge tube, and 2 ml of washed mitochondria containing 30-40 mg protein was layered carefully on top of the Percoll mixture and cen- trifuged f or 60 minutes at 48,000 g using angle rotor (in Sorvall RC
2B centrifuge). For the detection of the location of mitochondria, the gradient was fractionated using peristhaitic pump and a fine cap- illary tube dipped carefully to the bottom of the gradient and fractions of 1 ml were collected using an automatic fraction collec- tor. The 1 ml fractions were assayed for succinate dehydrogenase activity and protein content. Once the band containing activity was located, in subsequent experiments this band was collected with a -35-
syringe whose tip has been bent and made up to 50 ml with standard wash medium. Mitochondria were then collected by centrifuging at
48,000 g for 3 minutes. All procedures were conducted at 0-4°C and protein contents were determined by Lowry method.
6. Determination of endogenous NAD* contents
NAD+ contents were measured by the method described by Klingen- berg (1974). Three millilitres of mitochondrial suspension containing
30 mg protein when using Jerusalem artichoke mitochondria and 20 mg protein when using mung bean mitochondria were used for the determin- ation.
Samples were deproteinized by adding 0.2 ml of 3 N perchloric acid per ml mitochondrial suspension while mixing thoroughly and pro-
tein was then removed by centrifuging at 11,000 g for 10 minutes.
Supernatant was removed carefully and 0.6 ml ^HPO^ was added to the
supernatant while cooling in ice. Samples were neutralized by adding
3 N K0H until the pH was 7.2, and centrifuged again at 11,000 g for
10 minutes to remove KCLO^ precipatated. The supernatant was used
for the determination.
Measurements were carried out spectrophotometrically using DW-2
spectrophotometer at 340 nm in 1 cm light-path cuvettes in a total volume of 2.02 ml. One ml of the sample was added to 1 ml of 0.1 M pyrophosphate buffer (pH 8.8). Then 0.01 ml ethanol was added, mixed
and the extinction was recorded (Ej). 0.01 ml of alcohol dehydrogen-
ase (1.2 mg/ml) was added, mixed and the final extinction was recor-
+ ded (E2). NAD contents were calculated from the equation:
AE x V 6.22 -36-
where V is the volume of the assay mixture (ml); AE = E2~EJ, and 6.22
is the extinction coefficient for NADH at 340 nm.
7. Determination of endogenous TPP contents
Thiamine pyrophosphate content was determined by method described by Airth and Forester (1970). To 3 ml mitochondrial suspension (con-
taining 20 mg protein) 0.24 ml of 1.53 M trichloroacetic acid was
added, and protein precipitated was removed by centrifuging at 11,000
g for 10 minutes. To 2 ml of supernatant, 3.4 ml of 1.53 M potassium
acetate and 0.2 ml alkaline ferricyanide solution (prepared by dilut-
ing 0.65 ml of 0.059 M K^Fe (CN)& to 10 ml with 7.5 M NaOH), was add-
ed. The contents were mixed well and left to stand for 5 minutes at
room temperature. After this period of time, 0.1 ml of 30% ^02 was
added. The reaction was then extracted in 10 ml of isoamylalcohol
(128°-129° fraction) by mixing thoroughly using vortex mixer. The
isoamylalcohol phase was siphoned off and dried with 6 g of anhydrous
sodium sulphate for 1 hour. A series of TPP standards were prepared
and processed simultaneously together with an appropriate blank. Two ml of the isoamylalcohol extract was used for the determination. The
fluorescence was activated at 385 nm and detected at 435 nm. This was achieved using Perkin-Elmer MPF-3 Fluorescence Spectrophotometer.
8. Purification of NAD+-linked malic enzyme
a. Isolation of mitochondria
Jerusalem artichoke mitochondria were used for the purification
of the enzyme. 14 kg of peeled tubers were processed in 14 L of
standard isolation medium except that the MOPS concentration was in-
creased to 0.05 M. Mitochondria were isolated according to the method -37-
described previously. Mitochondrial pellet obtained was suspended in sonication medium consisting of 0.05 M MOPS and 0.01 M DTT at pH 7.0.
b. Sonication
The mitochondrial suspension was sonicated at 20 KC using a Dawe
Soniprobe tuned to maximum output. Samples were kept in ice-bath and sonication was for 30 seconds, 7 times with 30 seconds intervals for cooling. Samples were then centrifuged at 48,000 g. The supernatant containing the enzyme was assayed for malic enzyme, malate dehydro- genase and protein using assay mixture of 75 mM TES (pH 7.0),5 mM + DTT and 7.5 mM NAD . The reaction was started by adding 10 mM malate and when malate dehydrogenase activity reached equilibrium, 1.5 mM
MnCl2 was added to start the malic enzyme. Malate and MnCl2 concen- trations were total concentrations added to the reaction mixture.
c. Ammonium Sulphate fractionation
The supernatant from the sonication was made 45% saturated with ammonium sulphate, at 4°C and then centrifuged for 15 minutes at
40,000 g. The supernatant was assayed for malic enzyme activity and then made 60% saturated to ammonium sulphate. Protein precipated was then collected by centrifuging at 40,000 g for 15 minutes and dissol- ved in the least amount of resuspending medium consisting of Q.05 M
MOPS, 5 mM DTT and 1 niM MgCl2 at pH 7.0 and assayed for malic enzyme activity and protein.
d. Gel filtration
This was carried on a column of sephacryl S-200 (2.2 x 40 cm).
The column was washed thoroughly with 0.05 M MOPS (pH 7.0) containing
10 mM DTT before applying the sample. Sample obtained from 14 kg tubers was processed on one column. The effluent was collected in -38-
5 ml fractions (using automatic fraction collector) after being moni-
tored by a recording ultraviolet sensor (UV-cord LKB), indicating the presence of protein. Fractions with activity were pooled together and assayed for activity and protein.
e. DEAE-Sephadex
The diluted enzyme from the previous column was applied to a column of Sephadex A-25, which had been pre-equilibrated with 0.5 M
tris-sulphate buffer (pH 7.0) and washed with the same buffer plus
10 mM DTT before use. Once the enzyme was bound to the A-25 the column was washed with 0.05 M MOPS plus 10 mM DTT. A step wise gradient of 0.03, 0.1 and 0.3 M ammonium sulphate was used to elute
the protein from the column. Fractions of 5 ml were collected and those containing activity were pooled together and assayed for activi-
ty and protein; then made 65% saturated with ammonium sulphate. The enzyme was collected by centrifuging at 40,000 g for 20 minutes and
the pellet obtained was dissolved in the least amount of resuspending medium. All chromatography steps were conducted at Q-4°C, and sol- utions were degassed and stored at the same temperature before use.
f. Assay of enzyme activity
NAD+-linked malic enzyme was. assayed by measuring the increase
in absorbance at 340 nm associated with NAD+ reduction in a reaction mixture containing: enzyme, 75 mM TES buffer (pH 7.0), 50 mM malate,
+ 8 mM MgCl2 (or 4 mM MnCl2), 5 mM DTT and NAD in a concentration of
0-5 mM, in a total volume of 1 ml. Concentrations of malate, MgCl2 and MnCl2 were calculated as free concentrations. This was achieved by first calculating the concentration of Mg-malate complex and Mn- malate complex using the equation below: -39-
Concentration of the complex = (free malate) x (free metal) Kd where Kj is the dissociation constant of the complex. K^ forMg-malate
= 28.2 and for Mn-malate = 5.75 (Sillen and Martell 1964). The total concentrations added were the free concentration plus the complex con- centration.
A Perkin-Elmer model 555 Spectrophotometer was used and 1 cm light-path cuvettes.
Unit of enzyme activity was defined as the amount of enzyme which catalyzes the formation of one micromole of NADH per minute using 6.22 as the milliraolar extinction coefficient at 340 nm. And the specific activity was expressed as unit per milligram protein. Protein con- tent was estimated from 280/260 ratio. Absorbance was measured at
400 nm to account for any scattering, and value obtained was sub- stracted from readings at 280 and 260 before calculation. Protein was measured in 0.05 M MOPS buffer at pH 7.0.
9. Purification of NAD+-linked malate dehydrogenase
a. Ammonium sulphate fractionation
Mitochondria were isolated and sonicated as described before.
The crude extract was brought to 60% saturation by adding solid ammonium sulphate. The precipitate was removed after one hour by centrifuging at 40,000 g for 15 minutes. The supernatant was adjust- ed to 87% saturation with ammonium sulphate and protein was collected by centrifuging at 40,000 g for 15 minutes. The pellet obtained was dissolved in the least amount of resuspending medium (0.05 M MOPS,
5 mM DTT and 1 mM MgCl9, pH 7.0). -40-
b. Sephadex S-200
The sample was applied to a column of Sephacryl S-200 which had been pre-equilibrated with 0.05 M MOPS buffer (pH 7.0). The column was developed with the same buffer and the peak fractions showing enzyme activity were pooled and assayed for activity and protein con- tent. The activity was found in 60 ml, which was concentrated using amicon concentrator (minicon-B15).
c. Sephadex G-25
The concentrated sample was applied to a G-25 column pre- equilibrated with 0.05 M Tris-HCl buffer at pH 8.5. The column was developed with the same buffer. Fractions of 5 ml were collected and those with enzyme activity were bulked together and assayed. Activity was found in 15 ml.
d. DEAE-Sephadex
Sufficient Sephadex A-50, equilibrated in 0.05 M Tris-RCl buffer at pH 8.5, was added in a batch to the sample to absorb the enzyme. The ion-exchanger was then washed on a Buchner funnel with the Tris-HCl buffer until no further protein was eluted. Then washed with the same buffer containing 0.05 M NaCl and no activity was released at this concentration. The enzyme was then eluted from the resin in a single wash with 0.05 M Tris-HCl buffer containing 0.3M
NaCl. The malate dehydrogenase was precipitated by adding ammonium sulphate to the concentration of 80%, then collected by centrifuging at 40,000 g for 30 minutes. The pellet obtained was resuspended in the least amount of 0.05 M Tris-HCl buffer.
All chromatography steps were conducted at 0-4°C and solutions were degassed and stored at the same temperature before use. -41-
e. Assay of malate dehydrogenase
During purification procedures, enzyme activity was assayed by following the decrease in absorbance at 340 nm, due to the oxidation of NADH in a reaction mixture containing: enzyme, 75 mM TES buffer
(pH 7.0), 0.2 mM NADH, 0.5 mM OAA in a total volume of 1 ml. The re- action was initiated by the addition of OAA. Perkin-Elmer 555 Spec-
trophotometer was used.
Unit of enzyme activity was defined as the amount which, catalyzes
the transformation of one micromole of NADH per minute using milli— molar extinction coefficient of 6,22. And the specific activity was expressed as unit per milligram protein. The protein level was esti- mated from the 280/260 ratio and this was measured in 0.05 M MOPS buffer.
For studies on response to varying substrate and cofactor con-
centrations, malate dehydrogenase was assayed in the forward direction by measuring the increase in E^q associated with NAD+ reduction. The
assay mixture contained the enzyme, 0.05 M Tris-HCl buffer (pH 8.5),
5 mM NAD, 10 mM glutamate, 2 yl of GOT (2 mg/ml) in a total volume of
1 ml. The reaction was initiated by the addition of appropriate con-
centration of malate. The relation between reaction rate and enzyme
concentration was found to be linear. Assays were carried out using
a Perkin-Elmer spectrophotometer model 555. When studying the effect + of different concentrations of malate, 5 mM NAD was used and malate
concentration was varied (0-50 mM). When studying the effect of + + NAD , 20 mM malate was used and NAD concentration was varied
(0-5 mM). -42-
RESULTS
1. General Characteristics of Jerusalem artichoke mitochondria
Malate, succinate and externally added NADH are oxidized by
Jerusalem artichoke mitochondria. Respiratory activities of mitochon- dria isolated from Jerusalem artichoke tubers are shown in table (1).
ADP/0 ratio observed with malate as a substrate were close to 3.0, but when externally added NADH and succinate were the substrates the ADP/0 ratios obtained were nearly 2.0, indicating that ATP synthesis was coupled to electron transport at two sites only. These values for the efficiency of ATP synthesis are comparable with those obtained by other workers (Srivastava and Sarkissian 1970; Day and Wiskich 1974b; Douce et al. 1972).
Isolated plant mitochondria differ from mammalian mitochondria in / that most of them oxidize malate rapidly in the absence of a system to remove oxaloacetate (Wiskich and Bonner 1963; Ikuma and Bonner 1967;
Sarkissian and Srivastava 1968; Macrae and Moorhouse 1970; Macrae 1971a;
Coleman and Palmer 1972; Brunton and Palmer 1973). Plant mitochondria are able to oxidize malate because they contain NAD+-linked malic en-
zyme (Macrae and Moorhouse 1970) which is not subject to inhibition by accumulation of oxaloacetate. Thus plant mitochondria oxidize malate via two pathways, one is via malate dehydrogenase giving oxaloacetate and another via malic enzyme giving pyruvate. Pyruvate can be decar- boxylated and dehydrogenated to yield acetyl-CoA which would be fur-
ther metabolized by condensation with oxaloacetate to give citrate.
In contrast mammalian mitochondria fail to oxidize malate in the ab-
sence of a system to remove oxaloacetate, because the only enzyme
responsible for the catalysis of malate oxidation is malate dehydro- -43-
nmoles ^/min/mg protein Substrate RCR ADP/0 State 3 State 4
NADH 153 53 2.9 1.6
Succinate 148 44 3.4 1.8
Malate 49 15 3.3 2.8
Table (1) Respiratory properties of Jerusalem artichoke mito-
chondria. Additions were: 1 ml standard reaction medium,
1 mg mitochondrial protein, 2 mM NADH, 10 mM succinate,
25 mM malate and 0.1 mM ADP. State 3 rate represents
oxygen uptake in the presence of ADP and State 4 rate
when ADP has been phosphorylated. RCR, respiratory
control ratio. -44-
genase (EC 1.1.1.37). In mammalian mitochondria, malate oxidation can only be achieved if the oxaloacetate produced is removed, since the re- —12 action equilibrium (K 2.3 x 10 ) favours malate formation from e nq oxaloacetate. Several methods are possible for the removal of oxalo- acetate, by transamination to aspartate with glutamate and GOT
(Hobson 1970), or by condensation with acetyl-CoA to form citrate
(Lance et al. 1967) in the presence of citrate synthase.
Fig. (1) shows the rate of oxygen uptake when mitochondria iso- lated from Jerusalem artichoke or rat liver, oxidize malate supplied at different concentrations. Jerusalem artichoke mitochondria oxidized malate at a reasonable rate, however the addition of glutamate together with the enzyme glutamate-oxaloacetate transaminase (GOT) or thiamine pyrophosphate (TPP), increased the rate of oxygen uptake (Fig. 3A and
B). In case of rat liver mitochondria the rate of malate oxidation was very low, the inclusion of glutamate together with the enzyme GOT in the system caused a large stimulation in the rate of oxygen uptake; the addition of thiamine pyrophosphate had no effect. The presence of glutamate and GOT relieved the inhibition caused by oxaloacetate, by transamination to aspartate. GOT Oxaloacetate + L-glutamate fc aspartate + 2-oxoglutarate
Thiamine pyrophosphate had no effect on the rate of malate oxidation by rat liver mitochondria, because thiamine pyrophosphate is believed to act as a cofactor for the decarboxylation of pyruvate and animal mitochondria do not contain a malic enzyme to produce the pyruvate.
On the other hand oxidation of malate by Jerusalem artichoke mito- chondria via malate dehydrogenase and malic enzyme gave oxaloacetate and pyruvate, and the addition of thiamine pyrophosphate would be expected to enhance the production of acetyl-CoA which could have the -IO Ln I
Fig. (1) Malate oxidation by Jerusalem artichoke mitochondria (A) and rat liver mitochondria (B). Oxygen uptake measured as described in Materials and Methods. Additions were: 0.5 mM ADP, malate (0-100 mM), (•—• ), 10 mM. glutamate and GOT —o ) or 0.4 mM TPP ). -46-
effect of lowering the concentration of oxaloacetate, thus increasing
the contribution that malate dehydrogenase makes to the overall rate of malate oxidation.
2. Pyruvate oxidation by Jerusalem artichoke mitochondria
Jerusalem artichoke mitochondria oxidized pyruvate with a very
low rate. When 1 mM malate was added to the system, pyruvate oxidation
started with a very slow rate and after a period of time, the rate in-
creased and approached a constant rate (Fig. 2B). Inclusion of malate
in the system served as a source for an acetyl-acceptor by providing oxaloacetate (Walker and Beevers 1956). The initial slow rate of oxi- dation might be attributed to the lack of TPP or to the slow rate of pyruvate entry into the mitochondria. It has been reported that rat liver mitochondria posses a relatively specific pyruvate transporter in the
inner membrane and that pyruvate enters in exchange for 0H~ (Wiskich
1977) generated by the charge separation caused by the process of
electron transport.
When thiamine pyrophosphate and a sparker concentration of malate were included in the reaction medium, pyruvate oxidation responded
immediately to the addition of ADP (Fig. 2A) showing a respiratory con-
trol of 3.6 and ADP/0 ratio approaching 3.0 (2.4-2.6). TPP is known to
be an essential cofactor for pyruvate dehydrogenase (Sanadi 1963).
Lance et al. (1967) showed that particles from early preclimacteric
avocado, oxidized pyruvate only when both TPP and sparker concentra-
tion of malate were present. Similar results were reported by Douce
et al. (1977) using spinach leaf mitochondria.
It seems that the slow rate of pyruvate oxidation is limited by
the rate of pyruvate transport as well as the availability of TPP.
For the reaction to proceed at its maximal rate both malate and TPP I -O
The effect of TPP on Fig. (2) Pyruvate oxidation by Jerusalem artichoke mitochondria. Additions to 1 ml standard reaction medium were: 15 mM pyruvate, 0.4 and 0.2 mM ADP, 1 mM malate (Fig. 2B) and 0.4 mM TPP (Fig. 2A), 0.9 mg mito- chondrial protein was used. The values under the traces are the rates of oxygen consumption in nmoles/min/mg protein. -48-
must be included in the reaction mixture. Malate is transported fas- ter than pyruvate (Day and Hanson 1977b), thus producing some pyruvate which would supplement that which is imported, and also produce oxalo- acetate which would serve as acetyl-CoA acceptor.
It was noticed that the rate of oxygen uptake in the presence of
TPP was constant (Fig. 2A) and about twice the magnitude of that in the absence of TPP (Fig. 2B). This conclusion lends support to the conclusion reached earlier that the stimulation of malate oxidation was caused by enhanced rate of pyruvate metabolism to acetyl-CoA.
3. Malate oxidation by Jerusalem artichoke mitochondria
When Jerusalem artichoke mitochondria oxidized malate, they showed a strong increase in the rate of oxygen consumption in response to the addition of ADP. However, the reaction had a particular feature, the rate of oxidation decreased progressively as the reaction proceeded
(Fig. 3A). When ADP was added in a small proportion and malate oxi- dation was made to go through repeated state 3 to state 4 (state 3 and state 4 as defined by Chance and Williams 1956) transitions, the inhibition by product was less severe, and the state 3 rate decreased with time but to a lesser extent. This suggests that when high con- centration of ADP was added and malate oxidation was made to go through a continuous state 3 an intermediate product (OAA) was formed faster than it was removed, thus causing inhibition of the overall reaction. While, when smaller concentration of ADP was added, and the reaction was made to go through state 3 to state 4 transitions) the oxaloacetate formed is probably removed during state 4 as the ATP accumulated during state 3. It was suggested that ATP stimulates the oxaloacetate removal during the oxidation of1 malate or succinate Fig. (3) Malate oxidation by Jerusalem artichoke mitochondria and the effect of cofactors. Additions to assay medium were 0.9 mg protein, 30 mM malate, 0.4 and 0.2 mM ADP, 10 mM glutamate plus 2 yl GOT (B), 0.4 mM TPP (C), and 1 mM NAD (D). Figures represent the rate of uptake/min/mg protein. -50-
(Hulme et al. 1967; Goonwardena and Wilson 1979a). Hobson (1970) ob- served inhibited rate before the transition to state 4 from state 3, and reported that the duration of this phase was proportional to the amount of added ADP.
a. The effect of thiamine pyrophosphate
Data presented in Fig. (3C) demonstrate the effect of TPP on the state 3 rate of malate oxidation. Thiamine pyrophosphate stimulated the rate of oxygen uptake, besides it seems to have relieved the in- hibition caused by the accumulation of oxaloacetate.
It is difficult to see how thiamine pyrophosphate could affect the rate of malate oxidation by directly affecting the activity of the malate dehydrogenase or malic enzyme, but could bring its bene- ficial effect by increasing the rate of conversion of pyruvate to acetyl-CoA thus resulting in a better integration between the activi- ties of the two enzymes responsible for the oxidation of malate and preventing an excessive accumulation of oxaloacetate.
Fig. (4) shows results obtained when Jerusalem artichoke mito- chondria oxidized malate at different concentrations in the absence and presence of TPP. It was noticed that TPP decreased the apparent
K^ for malate (Fig. 4A). In the absence of TPP the apparent K^ for malate was 7 mM, while in the presence of TPP the apparent k^ was
3.5 mM. It was also noticed that TPP increased the maximum velocity.
At high malate concentration it was observed that in the presence of
TPP, the rate of oxygen uptake decreased, this was probably due to the lower activity of malate dehydrogenase at excessively high malate concentration (Bernstein et al. 1978). I Ln I
Flg* ^ Effect of TPP and NAD+ on malate oxidation. Additions to assay medium were, malate (5-100 mM), 0.5 mM ADP (•—• ) and 0.4 mM TPP * ) or 1 mM NAD+ ( o—o ). Fig. (4A) represents the initial rates, Fig. (4B) the final rates. -52-
Fig. (5) Malate oxidation and the effect of NAD and TPP. Additions to assay medium were: 0.9 mg mitochondrial protein, (0-40) mM malate, 0.2 mM ADP (•— ), 1 mM NAD+ (o—o ), 0.4 mM TPP ) or 0.4 mM TPP + 1 mM NAD+ (a—a ). -53-
b. Effect of exogenous NAD ori malate oxidation
The rate of malate oxidation was stimulated by the addition of + ... NAD (Fig. 3D and 4). The stimulation was particularly apparent when + ... NAD was added sometime after the initiation of the reaction, when the
control rate had significantly declined due to the accumulation of ox-
aloacetate. The enhanced rate of oxidation under the oxaloacetate
limited conditions must be due to an enzyme that Is not subject to the
equilibrium impossed on the internal malate dehydrogenase. It could be an external malate dehydrogenase or malic enzyme whose activity is
limited by the availability of NAD*. + The stimulation of malate oxidation by exogenously added NAD was reported by many workers (Day and Wiskich 1974a,b; Palmer and Arron
1976; Wedding et al. 1976; Neuberger and Douce 1978). Neuberger and
Douce (1978) suggested that the stimulation of malate oxidation in-
duced by the addition of NAD+, was due to the Increased concentration
+
of NAD in the matrix, assuming that the mitochondrial inner membrane
is permeable to NAD*. Experiments done during this study are con-
sistent with the accepted view that the mitochondrial inner membrane
is impermeable to NAD* (results will be shown in the following section).
Von Jagow and Klingenberg (1970) showed that mitochondria isolated 14 + from yeast cells are impermeable to C NAD .
Coleman and Palmer (1972), Brunton and Palmer (1973),and Palmer
and Arron (1976),suggested that exogenous NAD* stimulated a component
of malic enzyme situated in the intermembrane compartment and the
NADH produced is reoxidized by a specific NADH dehydrogenase located
near the external face of the inner membrane. On the other hand, Day
and Wiskich (1974a,b) indicated that malate oxidation takes place in
the matrix and they proposed that a transmembrane transhydrogenase -54-
+ was responsible for the reduction of exogenous NAD and the resultant
NADH can be oxidized by the external NADH dehydrogenase.
The location of the enzymes responsible for the oxidation of mal- ate requires confirmation. As the n-butyl malonate used in the location studies has been reported to inhibit malate oxidizing enzyme
(Phillips and Williams 1973). Also the observation made by Neuberger and Douce (1978) that plant mitochondria posses NAD+ translocator made it necessary to investigate the location of malate oxidizing enzymes in
Jerusalem artichoke mitochondria.
Addition of 1 mM NAD+ relieved the piericidin A inhibition of malate oxidation (Table 2). Under these conditions it was noticed that the addition of NAD+ caused a stimulation of oxygen uptake when no substrate was supplied. The same observation was made by Palmer and Arron (1976) and they assumed that under these conditions a low level of endogenous substrate was present in the washed preparation which might have served as the electron donor. Palmer and Arron (1976) also reported that the addition of NAD decreased the apparent for malate.
Table (2) shows the rates of oxygen uptake when malate or citrate was supplied as substrate and the effect of added NAD+ on the rate of oxygen uptake after piericidin A addition. It can be seen from these results that the oxidation of malate was stimulated to a greater ex- tent than that of citrate. As the stimulation of citrate is not sig- nificantly greater than the control, it is likely that NAD+ is stimu- lating a reaction unique to malate and which is not generally appli- cable to all NAD+-linked substrates. These results suggest that in + . the presence of exogenous NAD , another pathway for the oxidation of malate was in operation besides the normal one (malate oxidation in -55-
oxygen uptake nmoles/min/mg protein Substrate + P/A + P/A + NAD+
no added Substrate 0 0 12 ± 0.5
Malate 57 ± 1.0 20 ± 0.7 50 ± 0.6
Citrate 36 ± 0.5 17 ± 0.5 32 ± 1.0
Table (2) Stimulation of oxygen uptake by exogenous NAD+. Oxygen
uptake was measured as described in Materials and Methods.
Other additions were 30 mM Malate or 10 mM citrate, 0.4 mM
TPP, 0.9 mg mitochondrial protein, 0.2 mM ADP, 20.8 ng + piericidin A (P/A) and 1 mM NAD when appropriate. -56-
+ ... the absence of NAD ). As the inner mitochondrial membrane has been assumed to be impermeable to added NAD+, it has been suggested that this pathway is associated with, the outer face of the inner membrane.
Palmer and Arron (1976) using Jerusalem artichoke mitochondria showed + that pyruvate was the main product of malate oxidation when NAD and piericidin A were present. It seems reasonable to assume that this component is malic enzyme.
c. Effect of piericidin A on malate oxidation
Addition of piericidin A to mitochondria oxidizing malate caused a severe inhibition (Fig. 6A), after a period of time a piericidin A- resistant rate was observed to develop. In this case it seems that piericidin A together with oxaloacetate, produced as a function of malate dehydrogenase, had a synergistic effect on the rate of malate oxidation. Inclusion of TPP in the reaction medium reduced the dur- ation of the lag before the onset of the resistant rate (Fig. 6B), thus indicating the role of oxaloacetate in causing the inhibition.
Also emphasises that TPP probably removes accumulated oxaloacetate as already assumed. This result suggests that if no precautions were taken to remove oxaloacetate the only dehydrogenase in operation was the piericidin A sensitive dehydrogenase, as piericidin A was found to cause almost complete inhibition of oxygen uptake in the ab- sence of TPP (Fig. 6A).
Addition of 1 mM NAD+ relieved the piericidin A inhibition and a rate almost equivalent to the original rate was attained. It has been shown that in the presence of added NAD+ malate oxidation can by-pass the piericidin A- (or rotenone) sensitive site of phosphory- lation and in this respect resembles exogenous NADH oxidation (Day and Wiskich 1974a, Coleman and Palmer 1972). Coleman and Palmer 0972) I I
Fig. (6) The effect of piericidin A on malate oxidation. Additions to assay medium were: 30 mM malate, 0.5 mM ADP, 20.8 ng piericidin A (P/A),
1 mM NAD (A) and 0.4 mM TPP (B). Figures represent the rate of 02 uptake/min/mg protein. -58-
and Brunton and Palmer (1973) attributed the effect of exogenous NAD+
to the activity of malic enzyme which requires NAD+ as a cofactor.
Because NAD+ stimulated the rate of oxygen uptake in the presence of oxaloacetate limited rate and also pyruvate was the major product under
these conditions, they suggested that this enzyme is located in the intermembrane space. In this case the reduced NAD+ will be reoxidized by a specific NADH dehydrogenase located near the external face of the inner membrane (Douce et al. 1973; Jagow and Klingenberg 1970). In transhydrogenas e contrast Wiskich and Day (1979) suggested that a transmembrane/may be involved in the transfer of reducing equivalents from the internal
NADH in matrix space to the external NAD+. Neuberger and Douce (1978) claimed that plant mitochondria possess an NAD+-translocator.
For a further investigation of the location of malic enzyme, n- buytlmalonate was used, and the effect of this compound on the oxi- dation of malate and the NAD+-stimulated rate was studied.
d. The effect of n-butylmalonate on malate Oxidation
Exchange carriers for the transport of substrate anions exist in
the inner membrane of animal (La Noue and Schoolworth 1979) and plant
(Wiskich 1975; Day and Hanson 1977a) mitochondria. Dicarboxylic anions such as malate and succinate enter in exchange for phosphate via a carrier which can be inhibited by substrate analogous such as 2-n-butyl- malonate (Phillips and Williams 1973; Robinson and Chappell 1967;
Robinson et al. 1972; Johnson and Chappell 1973). Results obtained in this study showed that butylmalonate inhibited the state 3 rate of malate oxidation by Jerusalem artichoke mitochondria (Fig. 7). At a concentration of 15 mM butylmalonate, the rate of oxygen uptake was
inhibited by 49% when 10 mM malate was used as a substrate. On the other hand, NAD+-stimulated rate only was slightly affected (14%) by -59-
Fig. (7) The effect of n-butylmaloiiate on the rate of malate oxidation in the presence and absence of 1 mM NAD*. Additions to assay medium were 10 mM malate, 0.4 mM TPP ( •—• ), 20.8 ng piericidin A and 1 mM NAD* ° ). -60-
butylmalonate over the same range of concentrations used. In this experiment TPP was included in the reaction meduim to eliminate any inhibition caused by the accumulation of oxaloacetate. When studying the effect of butylmalonate on the NAD+-stimulated rate, piericidin A was included in the reaction mixture.
Another experiment was carried out in which mitochondria were allowed to oxidize 50 mM malate in the presence of varying concentrat- ions of butylmalonate, then piericidin A was added. When piericidin
A-resistant rate was attained, 1 mM NAD+ was added. In this experi- ment piericidin A-resistant rate was found to be inhibited by butyl- malonate. When this rate was substracted from the rate following + . . + NAD addition, the NAD -stimulated rate proved to be almost constant
(Fig. 8 and table 3), and unaffected by the presence of butylmalonate.
In the experiment described above, butylmalonate may be prevent- ing the entry of malate or inhibiting the enzyme responsible for the oxidation of malate (Phillips and Williams 1973; Palmer and Arron
1976). An experiment using disrupted mitochondria was carried out to test this possibility and results obtained (table 4) showed that buty— lmalonate had no effect on the enzyme within the range of concentrat- ions used. Butylmalonate at a concentration of 15 mM had no effect on malate dehydrogenase activity, while it inhibited the rate of oxygen uptake by 49% (in the absence of added NAD+). Day and Hanson
(1977a) using corn mitochondria reported that butylmalonate had no effect on malic dehydrogenase.
Coleman and Palmer (1972) and Palmer and Arron CI 976) showed that + in the presence of piericidin A and added NAD , the product of malate oxidation was mainly pyruvate. In this study n-butylmalonate was found to have no effect on the NAD+-stimulated rate of malate Butyl malonate State 3 rate NAD-stimulated rate concentration nmoles 02/min/ % Inhibition + P/A + NAD* nmoles 02/min/mg mM mg protein protein
0 33 ± 0.7 0 8.0 ± 0.4 31 ± 1.0 23
6 28 ± 0.9 15 5.0 ± 0.2 27 ± 0.8 22
12 23 ± 0.4 30 4.0 ± 0.2 27 ± 0.7 23
18 20 ± 1.0 33 4.0 ± 0.4 27 ± 0.6 23
30 14 ± 0.4 57 2.0 ± 0.3 26 ± 0.9 24
Table (3) Effect of n-butyl malonate concentration on malate oxidation by Jerusalem artichoke
mitochondria. Additions were 50 mM malate, OA mM TPP, 0.2 mM ADP, 20.8 ng piericidin A
(P/A) and 1 mM NAD. Results are mean of 5 readings ± S.E. -62-
40 -1
e
l 30 - To» E O —1 c* ( 'S 20-
~o E c 10-
00 6 12 16 ik 3b mM n-butylmalonate
Fig. (8) The effect of n-butylmalonate on the rate + of malate oxidation and the NAD -stimulated rate. Assay was carried in 1 ml reaction medium, additions were 50 mM malate, 0.4 mM TPP ( —• ), 20.8 ng piericidin A and 1 mM NAD+ 15 ). -63-
Rate of NADH oxidation
ymole/min/mg protein
Control + 15 mM BM
29769 ± 277 29265 ± 210
Table (4) The influence of butyl malonate (BM) on malate de-
hydrogenase activity. Malate dehydrogenase was assayed
by monitoring the oxidation of NADH in the presence of
oxaloactate (OAA). The reaction was carried out in 1 ml
MES buffer (pH 6.8), O.l^of triton X 100, 50 ng Antimycin
A, 0.15 mM NADH and 0.3 mM OAA. Rates are mean of 4
readings ± S.E. -64-
oxidation (Fig. 8). These observations suggest that malate need not be transferred through the inner membrane to be oxidized under these conditions. It might be concluded from these results that in the . . . + presence of piericidin A and exogenous NAD malate oxidation takes place in the intermembrane compartment and the NADH produced is re- oxidized by the NADH dehydrogenase located on the outer surface of the inner membrane.
e. Effect of oxaloacetate on malate oxidation
Hulme et al. (1967) studying the oxaloacetate metabolism in apple mitochondria, suggested that oxaloacetate was decarboxylated directly to pyruvate and that the NADH and acetyl-CoA produced as a result of pyruvate oxidation were used to further reduce the concen- tration of oxaloacetate. The same system for the removal of oxalo- acetate was proposed by Avron and Biale (1957). Avron and Biale
(1957) demonstrated that the NADH produced by pyruvate oxidation could be used to reduce oxaloacetate while NADH produced by the oxidation of malate continuedto be oxidized by the respiratory chain.
Many workers have observed that added oxaloacetate readily enters plant mitochondria and exerts a powerful, but transient inhibition of the rate of oxidation of most Krebs-cycle dehydrogenases (Avron and
Biale 1957; Hulme et al. 1967; Douce and Bonner 1972; Brunton and
Palmer 1973). Douce and Bonner (1972) showed that oxaloacetate added to mitochondria respiring under state 4 conditions caused a transient inhibition of oxygen uptake accompanied by the complete oxidation of the pyridine nucleotide pool. Then the NAD* became reduced when the oxaloacetate had been removed and the rate of oxygen uptake recovered.
They concluded that the NADH produced by the malic enzyme was diverted from the respiratory chain to reduce the oxaloacetate. -65-
Brunton arid Palmer (1973) using wheat mitochondria observed that
when oxaloacetate was added to mitochondria respiring under state 3
conditions, in the presence of pyruvate or citrate, it caused a power-
ful but transient inhibition of the rate of oxygen uptake; whilst a
partial but permanent inhibition was observed when malate was the sub-
strate. They suggested that under state 3 conditions, NADH produced
by the malic enzyme was not freely available for the reduction of oxa-
loacetate and not diverted from the respiratory chain.
Fig. (9) shows the rate of citrate oxidation and the effect of
added oxaloacetate to mitochondria respiring under state 3 conditions.
Addition of oxaloacetate caused a powerful but transient inhibition
of citrate oxidation (Fig. 9B). It seems that under these conditions
NADH produced by citrate oxidation was used to reverse the function
of malate dehydrogenase (i.e. conversion of oxaloacetate to malate).
And when the oxaloacetate was lowered to the equilibrium concentration,
the inhibition of oxygen consumption was released.
When malate was used as a substrate, the rate of oxygen uptake
tended to slow down with time (Fig. 10A); this is presumably due to
the accumulation of oxaloacetate produced as a result of malate de- hydrogenase activity. When oxaloacetate was added to mitochondria
oxidizing malate under state 3 conditions, it caused a partial inhib-
ition and the rate of oxygen uptake tended to recover but never
attained the initial rate (Fig. 10B). However, this rate was almost
equivalent to the final rate achieved by malate oxidation without the
addition of exogenous oxaloacetate (Fig. 10A).
When thiamine pyrophosphate was included In the reaction mixture,
oxaloacetate caused partial inhibition and a rate almost equivalent
to the initial rate was attained after a short time (Fig. 1QC). OIn ON I
Fig. (9) Influence of OAA on State 3 rate of citrate oxidation. Initial concentrations of additions are: 15 mM citrate, 0.66 mM ADP, 0.2 mM oxaloacetate (OAA). Figures represent the rate of 0„ uptake/min/mg protein. I ON I
Fig. (10) Influence of OAA on State 3 rate of malate oxidation. Initial concentrations of additions are: 30 mM malate, 0.66 mM ADP, 0.2 mM OAA (B), 0.4 mM TPP (C), 0.4 mM TPP and 1 mM NAD (D). Figures represent the rate of uptake/min/mg protein. -68-
+ . Addition of NAD increased the rate of oxygen uptake and shortened
the time needed for the rate of oxygen uptake to recover (Fig. 10D).
Results obtained in this study showed that malate oxidation was + stimulated by exogenously added NAD . Malate oxidation was inhibited by piericidin A and this inhibition was relieved upon addition of 1 mM + ..... NAD . It was also observed that malate oxidation was inhibited by n- + butylmalonate, while the NAD -stimulated rate was insensitive to this
inhibitor, suggesting that in the presence of piericidin A and exter- nally added NAD+, malate oxidation was independent of malate translo-
cation through the inner mitochondrial membrane. In addition malate
oxidation in the presence of piericidin A was stimulated to a greater
extent by added NAD* than the oxidation of citrate. From these re-
sults, it was suggested that NAD* added externally was not acting as
a substrate for a transmembrane transhydrogenase (Day and Wiskich
1974a,b) and probably stimulating a component of malate oxidizing
system present in the intermembrane compartment of the mitochondria.
By differential centrifugation, isolation yield preparation of organelles which are adequate for the study of electron transport pathway and coupling processes. These preparations are less suitable
for studies of enzyme distribution. Studies concerning the location of enzymes with respect to the membrane system require very pure and
intact mitochondria. Thus it was necessary -to investigate methods
for improving the purity and integrity of the final mitochondrial preparation. These studies coincided with Percoll becoming available .
4. Purification of mitochondria
Procedures using continuous and discontinuous sucrose gradients have been developed to purify mitochondria isolated by the usual dif-
ferential centrifugation (Douce et al. 1972; Day and Hanson 1977c, -69-
Douce et al. 1977). Another procedure for purifying mitochondria was by partition in an aqueous dixtranpoly-(ethylene glycol) two-phase system (Gardestrorn et al. 1978). In this system particles are dis- tributed according to their surface properties (Albertsson 1974).
Bergman et al. (1980) developed a three-step procedure for preparing mitochondria from spinach leaves. Their procedure involved, differen- tial centrifugation, partition in an aqueous dextran-PEG two phase system and Percoll gradient. The final preparation from the Percoll density gradient was totally free of chlorophyll.
The use of Percoll which is a silica sol having low viscosity, low osmolarity and non-toxic, made it possible to avoid the lengthy procedure and the stress the organelles suffer during the dilution to isosmoticconcentration when using sucrose gradient. Jackson et al.
(1979) described a technique for purifying mitochondria from green leaves. A three-step discontinuous gradient of Percoll was used. By this method, they could remove most of the chlorophyll from the washed mitochondria which retained intactness, respiratory control and ADP/0 ratio.
For the purification of mitochondria Percoll mixture was used in different concentrations (15, 20, 25% v/v) and 20% (v/v) proved to be suitable concentration. Using 20% (v/v) Percoll mixture, different timings were used and 60 minutes was found to be the most effective timing.
Washed mitochondrial preparation was layered on top of self- generating gradient (20% (v/v) Percoll mixture), and after centrifug- ing for 60 minutes, mitochondria were found to form a single band about 1 cm above the bottom of the centrifuge tube in case of potato and mung bean mitochondria. In the case of Jerusalem artichoke mito- chondria using the same concentration of Percoll (20%) mitochondria -70-
were found in two bands, a major lower band, which was about 1.5 cm
above the bottom of the centrifuge tube and another one about 3 cm
above the lower band. When examining the quality of the mitochondria,
it proved that mitochondria of both bands were of good quality. This
might indicate that there are two populations of Jerusalem artichoke
mitochondria. As the purpose of this study was to achieve intact
mitochondria a lower concentration of Percoll was used to recover
mitochondria in a single band. When using 18% (v/v) Percoll mixture,
mitochondria were recovered in a single band, although it was wider
than that associated with potato or mung bean mitochondria.
In all cases protein containing material was found at the bottom
of the centrifuge tube and greenish material of high protein content
was found at the top of the gradient which contained no mitochondrial
activity.
Percoll mixture was prepared by making up 20% (v/v) Percoll in
standard wash medium, 40 ml of this mixture was placed in a 50 ml
centrifuge tube and centrifuged for 60 minutes. The gradient formed
was fractionated into(Mml fractions and the refractive index for the
O^ml fractions was measured; values obtained were drawn against the
number of fractions (Fig. 11). The density of the fractions were
calculated from the standard curve supplied by Pharmacia 0977).
Jerusalem artichoke mitochondria were found to be located at density
(1.095 g/ml) while potato and mung bean mitochondria were found at
density (1.098 g/ml). The profile of protein distribution in the fractination of the
gradient for material used in this study are illustrated in Fig.
(12-15).
Fig. (12) shows the distribution of Jerusalem artichoke mito- chondria detected by the presence of succinate dehydrogenase (SDH) i
Fractions (0 9 ml.) i
Fig. (11) Development of gradient of Percoll during centrifu- gation. Starting density 1.084 g/ml. 40 ml of 20% Percoll centrifuged for 60 min at 48,000 g. (T) top of gradient, (B) Bottom of gradient. I -vj N3 I
Fig. (12) Distribution of Jerusalem artichoke mitochondria on 20% Percoll density gradient. Mitochondria obtained from differential centrifugation were loaded on top of 20% self generating Percoll gradient and centrifuged for 60 min. -73-
activity, after the fractionation of a 20% Percoll gradient. The activity of succinate dehydrogenase was measured spectrophotometri- cally by the phenazine methosulphate-mediated reduction of DCIP at
600 nra. It can be seen from this figure that mitochondria are found in two bands. When using lower concentrations of Percoll (18%), mito- chondria were found in a single band which coincided with a major protein peak (Fig. 13).
Fig. (14 and 15) show distribution of mitochondria isolated from potato and mung bean hypocotyls. They show a similar pattern of dis- tribution of succinate dehydrogenase activity and protein contents as reported for Jerusalem artichoke mitochondria on an 18% Percoll gradient (Fig. 13).
a. Integrity of mitochondrial preparations
The integrity of mitochondrial preparation can be evaluated by measuring the integrity of the outer membrane, ADP/0 and respiratory control ratios. The integrity of the outer membrane of mitochondria isolated from Jerusalem artichoke, mung bean hypocotyls and potato tubers was measured by the activity of succinate-cytochrome c reduc- tase, which is considered to be located on the external face of the inner membrane. Cytochrome c does not pass through the outer membrane
(Wojkczak and Sottocasa 1972), and any succinate-cytochrome c reductase activity would indicate the damage of the outer membrane allowing external cytochrome c to reach its site of reduction located on the outer surface of the inner membrane.
Table (5) shows the rate of succinate-cytochrome c reductase activity in washed and purified preparations. When the osmolarity of the reaction medium was sufficient to prevent the bursting of the mitochondrial outer membrane, the rate of reduction was low in both (13) Distribution of Jerusalem artichoke mitochondria on 18% Percoll gradient. Solid line represents mitochondrial activity and broken line represent protein distribution. I
I Ln I
Fig. (14) Distribution of mung bean mitochondria on 20% Parcoll gradient. Solid line represent mitochondrial activity, broken line represent protein distribution. (15) Distribution of potato mitochondria.on 20% Percoll gradient. Solid line represent mitochondrial activity and broken line represent protein distribution. -77-
Mitochondria nmoles cyt c/min/mg .protein
Type Intact Osmotical as percentage intact Sourc e disrupted
Washed 41±0.6 444±15 91%
Purified 17±1.0 635±34 97% Artichok e
Washed 21±0.3 190± 4 89% bea n
Mun g Purified 24±3 327± 5 93%
Washed 41±1.5 360±12 89%
Potat o Purified 33 ±1.3 408±15 92%
Table (5) Cytochrome c reduction by mitochondria isolated from
Jerusalem artichoke, mung bean and potato. The assay
was carried out in 1 ml standard reaction medium using
20-40 pg mitochondria protein. Additions were; 3 mM
KCN, 0.05 mM cytochrome c and the reaction was initiated
by the addition of 10 mM succinate. -78-
washed and purified preparations. Bursting the mitochondria in a medium the osmolarity of which was approaching zero, resulted in the maximal rate of cytochrome c reduction. From the ratio of the rate of cytochrome c reduction in the intact mitochondria and that obtained after bursting, it can be calculated that the original preparation had between 89-91% intact outer membrane and that this value rose to between 92-97% intact outer membrane after purification. These results show that both washed and purified preparations are of acceptable quality and that the purified are significantly better than the washed preparation.
Table (6) gives average values for rates of oxidation, ADP/0 and respiratory control ratios with NADH as a substrate for washed and purified mitochondria. The data show that the state 3 rate is higher
in the purified than that in the washed mitochondria. In addition
ADP/0 and respiratory control ratios are higher. It was also observed
that the rate of NADH oxidation in the purified mung bean mitochondria was doubled over that of the washed preparation, while that in
Jerusalem artichoke mitochondria was increased by 50%. This suggests
that mung bean mitochondria benefit from purification more than
Jerusalem artichoke mitochondria.
On the basis of these criteria, mitochondria purified through continuous Percoll gradient were largely intact and the technique
seems comparable to sucrose gradient (Douce et al. 1972, Day and
Hanson 1977c), and has the advantages of avoiding the osmotic stress
that the organelles suffer upon purification through sucrose gradient.
The method needs less time, which can be in itself a factor in maintaining the integrity of isolated mitochondria. Day and Hanson
(,1977c) and Arron et al. Q979) reported a loss in respiratory control
through purification using sucrose gradient. -79-
nmoles Cty/min/mg protein Type of RCR ADP/0 Mitochondria State 3 State 4
Jerusalem artichoke
Washed 312 146 2.1 1.4
Purified 437 125 3.5 1.7
Mung bean
Washed 157 59 2.7 1.3
Purified 307 97 3.2 1.5
Table (6) NADH oxidation by washed and purified mitochondria isolated
from Jerusalem artichoke and mung bean. Assay was carried
out in 1 ml standard assay medium and additions were, 2 mM
NADH and 75 yM ADP. -80-
b. Malate oxidation
When studying the effect of cofactors on the rate of oxygen up- take, one must be aware of soluble enzymes released from broken mito- chondria, as their activity in the supernatant solution will be limited by the lack of coenzyme which will become excessively diluted In the supernatant medium. When adding NAD+, the concentration of this co- enzyme would be increased in the reaction mixture and would trigger the activity of the soluble enzyme.
Purification reduced broken mitochondria from 10% to 3%; there- fore, soluble enzymes in the purification is expected to be present in a very small concentration. If the response to added NAD+ was due only to the activity of soluble enzyme, then the response of purified mitochondria to added NAD+ would be expected to be very low. However, the addition of NAD+ to purified Jerusalem artichoke mitochondria oxidizing malate stimulated the rate of oxygen uptake significantly.
Table (7 and 8), show rates of malate oxidation in both washed and purified mitochondria isolated from Jerusalem artichoke tubers and mung bean hypocotyls, and the effect of added NAD+ and thiamine pyro- phosphate.
Table (7), shows that state 3 rate of malate oxidation in puri- fied mitochondria was slightly lower than that in washed mitochondria in the case of Jerusalem artichoke. In both washed and purified mitochondria the successive state 3 rates were lower than the first state 3, this presumably due to the accumulation of oxaloacetate.
The addition of NAD+ stimulated the rate of oxygen uptake in both washed and purified mitochondria, and the successive state 3 rates were maintained at the same level. This indicates that the addition of NAD+ stimulated a component of malate oxidizing system Malate oxidation nmo les 02/min/mg protein
Type of Addition State 3 State 4 State 3 State 4 State 3 State 4 State 3 ADP/0 Mitochondria
Malate 136 49 133 45 125 44 123 2.5
+ NAD+ 153 67 153 63 153 59 157 2.0
+ TPP 181 92 233 95 252 95 265 2.0 Washe d
+NAD + TPP 191 99 237 109 267 105 286 2.1
Malate 128 52 128 42 115 31 101 2.4
X) +NAD+ 158 60 172 49 175 46 193 2.1 0) •H CM *H +TPP 175 70 235 77 277 77 280 2.3 u p +NAD + TPP 262 98 322 98 322 98 267 2.1
Table (7) The effect of NAD+ and TPP on the rate of malate oxidation by washed and purified mitochondria
isolated from Jerusalem artichoke. Additions to the assay medium were: 30 mM malate, 0.4 mM
TPP, 1 mM NAD+, 60 yM of ADP was added to initiate the state 3 rate. Malate oxidation nmoles O^/min/mg protein
Type of Addition III IV III IV III IV III ADP/0 Mitos.
Malate 102 31 119 39 122 38 123 2.0
+ NAD+TPP 112 48 127 49 127 49 127 1.9 i
Malate 189 71 260 59 273 59 256 2.0
+ NAD* 218 80 265 71 281 67 260 2.1 id •H -- 4-1 •UH + TPP 210 76 281 84 294 84 328 2.2 P3M +NAD+TPP 210 76 273 84 294 84 311 2.1
Table (8) The effect of NAD and TPP on the rate of malate oxidation by washed and purified mito-
chondria isolated from mung bean hypocotyls. Assay was carried in 1 ml of standard assay
medium and additions were, 30 mM Malate, 0.4 mM TPP, 1 mM NAD* and 60 yM ADP was added to
initiate state 3 rate. -83-
as well as facilitating the removal of oxaloacetate. As the purified + preparation was highly intact, the added NAD was assumed to stimulate a component of malate oxidizing system present In the inter membrane compartment.
The addition of thiamine pyrophosphate increased the rate of oxy- gen uptake in both washed and purified mitochondria. The effect of
TPP was by eliminating the accumulation of oxaloacetate, by increasing the rate of acetyl-CoA formation from pyruvate, which would condense with oxaloacetate to give citrate. The successive state 3 rates were higher than the first state 3 rate in both preparations.
When TPP was included in the reaction medium to maintain con- + stant rates, addition of NAD stimulated further the rate of oxygen uptake in both the washed and purified mitochondria.
Table (8) shows rates of malate oxidation in washed and purified mung bean mitochondria. Rates of oxygen uptake were higher in the purified than in washed mitochondria. These results differ from those obtained with Jerusalem artichoke mitochondria (Table 7). + Addition of NAD only caused a slight increase m the rate of oxygen uptake and the addition of thiamine pyrophosphate had more effect on purified than on washed mitochondria.
c. Endogenous NAD+ and TPP contents
Neuberger and Douce (1978) reported that plant mitochondria possess an NAD+-transporting system and that the addition of NAD+ would . + increase the concentration of internal NAD , thus stimulating the rate of malate oxidation.
NAD+ contents were determined for Jerusalem artichoke and mung bean mitochondria for both washed and purified preparations and re- + suits are shown in Table (9). Results are expressed in nmoles NAD -84-
Source NAD+ contents nmoles/mg Protein TPP ug/mg P of Mitos. Washed Mitos. Purified Washed
Jerusalem 1.01 ± 0.07 1.5 ± 0.08 9.6 ± 0.6 artichokes
Mung beans 1.7 ± 0.14 3.4 ± 0.07 23.4 ± 0.4
Table (9) Endogenous NAD+ and TPP contents in Jerusalem + artichoke and mung bean mitochondria. NAD was
determined for washed and purified mitochondria.
TPP content was measured for washed mitochondria.
Values are mean of 10 experiments ± SE for NAD+
and mean of 5 experiments for TPP ± SE. -85-
per milligram protein and values are average of ten experiments. It + can be seen from results in Table (9) that NAD contents are higher in mung bean mitochondria than that of Jerusalem artichoke mito- chondria and values increased upon purification indicating the removal of non-mitochondrial protein and that NAD+ is not lost through puri- fication. Suggesting that the inner membrane is impermeable to NAD+.
The content of thiamine pyrophosphate was also determined for washed mitochondria from Jerusalem artichoke and mung bean and values are shown in Table (9). Levels of TPP are expressed as yg TPP per milligram mitochondrial protein. Mung bean mitochondria were found to contain more thiamine pyrophosphate than Jerusalem artichoke mito- chondria. This might explain why mung bean mitochondria respond less to the addition of thiamine pyrophosphate. + d. Permeability to NAD
The suggestion of Neuberger and Douce (1978) that NAD+ enters the mitochondria and accumulate in the matrix space but does not leave the matrix, suggests that it is possible to load the mitochondrial matrix by incubating the mitochondria with NAD+, then wash the mitochondria to eliminate any accumulation in the intermembrane space. In this case the addition of NAD+ would have no effect on malate oxidation.
When Jerusalem artichoke mitochondria were incubated with 2 mM
NAD+ at 4°C or 22°C for 5 minutes, then washed twice with regular wash + medium, the values obtained for NAD contents were not significantly higher than the control (Table 10). Values obtained were of the same magnitude as those obtained for purified mitochondria (Table 9). This presumably was due to the removal of non-mitochondrial protein upon washing. In both the control and treated mitochondria, the addition of 1 mM NAD+ stimulated the state 3 rate of malate oxidation, Table (10). Control Mitos. incubated with2mM NAD*
nmoles 02/min/mg P nmoles 02/min/mg P Additions NAD* NAD* State 3 State 4 State 3 State 4 content content nmoles/mg P nmoles/mg P
Malate 40.0 11.0 47.0 12.0
1.3±0.07 1.4 ±0.01
Malate ++ 57.0 17.0 57.0 16.0 1 mM NAD
+ + Table (10) NAD uptake by Jerusalem artichoke mitochondria, and the effect of NAD on the
rate of oxygen uptake. Mitochondria were incubated with 2 mM NAD* at 22°C,
then washed twice. Oxygen uptake was measured as described in Materials and
Methods Section. -87-
From these results it was suggested that Jerusalem artichoke mitochon- dria are impermeable to added NAD+. It was shown previously that in the presence of n-butyl malonate, the NAD+-stimulated rate of malate oxidation was independent of malate transport through the inner mem- brane. Thus this component was assumed to be present in the inter- membrane compartment.
e. Reduction of external NAD+ by Jerusalem artichoke mitochondria
Day and Wiskich (1978) using cauliflower mitochondria, observed that in the absence of oxaloacetate removing system, NAD+ reduction started with a fast rate which slowed to zero as the reaction pro- gressed. They also reported that this rate was dependent on inorganic phosphate and the removal of oxaloacetate produced. They attributed the reduction of exogenous NAD+ to the activity of malate dehydrogen- ase present in the matrix space and the slowing rate as a result of accumulating oxaloacetate and NADH in the matrix, which would equili- brate the reaction.
Intact Jerusalem artichoke mitochondria reduce exogenous NAD+ in a biphasic manner (Fig. 16). The initial rate of NAD+ reduction was fast and it tended to decrease to a slower and almost constant rate when using washed mitochondria. When using purified mitochondria, the initial rate was lower than that observed with washed mitochondria, while the final rate was higher. Data are shown in Table 01) and re- sults are expressed as nmoles NAD+ reduced per minute per milligram protein. + Unlike malate, citrate failed to induce external NAD reduction in the presence of antimycin A In both washed and purified mitochon- dria (Table 11). Similar results were obtained by Von Jagow and
Klingenberg (1970) and Day and Wiskich 0978). Palmer and Arron 0i0 00 I
Time
Fig. (16) Exogenous NAD* reduction by.washed (A) and purified (B) Jerusalem artichoke mitochondria. NAD* reduction at 340-373 nm in a reaction mixture containing: 1 ml standard reaction medium, 1 mM NAD*, 500 ng antimycin A and 10 mM malate. Figures represent ymol NAD* reduced/min/mg protein. -89-
Rate of NAD+ reduction nmoles/min/mg protein Type of Mitos. Substrate Initial rate Linear Rate
Washed Malate 90 ± 5 15 ± 0.3 Mitos.
Malate + 118.0 17.0 TPP
Citrate 0 0
Purified Malate 62 ± 2 19.0±1.0
Malate + 60.0 17.0 TPP
Citrate 0 0
Table (11) Reduction of exogenous NAD+ by intact Jerusalem artichoke
mitochondria. Assay was carried in 1 ml standard reaction
medium containing 1 mM NAD+, 0.4 mM TPP, 500 ng Antimycin
A, the reaction was initiated by the addition of 10 mM
malate or 10 mM citrate. -90-
+ reported that citrate reduced exogenous NAD with very low rate compared to malate.
When thiamine pyrophosphate was included in the reaction medium to eliminate the accumulation of oxaloacetate, the initial rate of
NAD* reduction was increased when using washed mitochondria and the final steady rate was only slightly increased. From this result, the initial rate of reduction is probably due to the activity of malate dehydrogenase, while the final steady rate was a result of malic en- zyme activity. When using purified mitochondria inclusion of thiamine pyrophosphate had no effect on either the initial or the final rate.
From these results it is possible to conclude that the first initial rate of NAD* reduction was probably due to malate dehydrogenase released from broken mitochondria. Because malate dehydrogen- ase is very active enzyme, the release of 3% of the total malate de- hydrogenase give a good rate if NAD* is added. As Jerusalem artichoke mitochondria were found to contain a much higher component of malate dehydrogenase in the matrix, than malic enzyme (results will be shown in the following section).
From results mentioned in the previous section, citrate oxidation in the presence of piericidin A was found to be stimulated by exogen- ous NAD* to much lower extent than the rate of malate oxidation
(Table 2). Citrate was also found to be incapable of reducing exo- genous ly supplied NAD*. If a transmembrane transhydrogenase was in- volved in transferring reducing equivalents from endogenous NADH to
+ + external NAD , all NAD -linked substrates would be expected to induce external NAD* reduction. In addition n-butyl malonate was found to + have no effect on NAD -stimulated rate of oxidation when malate was used as a substrate (Table 3). These observations are not consis- tent with the operation of transhydrogenase. -91-
Neuberger and Douce (.1978) and Tobin et al. (1980) suggested that plant mitochondria posses an NAD+ transporting system. And reported
that malate oxidation in potato mitochondria which contain low level of endogenous pyridine nucleotide, respond to added NAD+, while mung bean mitochondria which contain relatively higher level of endogenous pyridine nucleotide showed less response to added NAD+. Suggesting
that in the case of potato mitochondria, the level of endogenous NAD+ was too low to saturate the malic enzyme, which was activated when
NAD+ was added.
An attempt was made to isolate and purify enzymes responsible
for the oxidation of malate and some of their kinetic properties were
studied.
5. Purification of enzymes responsible for malate oxidation
a. Purification of NAD+-malic enzyme
The malic enzyme was isolated and purified from mitochondrial preparation as described in "Materials and Methods" section. An in-
crease of specific activity of 126 fold over the level of the sonic
supernatant obtained from mitochondria was achieved. A flow sheet
of the purification is shown in Table (12) and the elution profile
of protein and enzyme activity from the sephacryl S—200 gel filtration
column is given in Fig. (17A) and from Sephadex A-25 column in Fig.
(17B). A large quantity of inactive protein was eluted from the
Sephadex A-25 column by 0.01 M and 0.03 M ammonium sulphate before
the elution of the enzyme by 0.3 M ammonium sulphate (Fig. 17B).
Sonicated mitochondrial supernatant was used as a reference to cal-
culate the relative purification and protein recoveries. The final
preparation has a specific activity of 9.9 unit per milligram protein ) • • • Total volume Total activity Total protein Specific activity Stage ml ymole NAD+/min mg unit/mg protein
Sonic supernatant 220 330.0 4203.0 0.0785
45-60% AS 2.5 237.5 332.5 0.714
After S-200 24.0 77.17 84.0 0.918
After A-25 15.0 35.0 7.8 4.5
Final preparation 0.6 10.6 1.07 9.9
Table (12) Purification of malic enzyme from Jerusalem artichoke mitochondria. Assays were
carried out at pH 7.0, at room temperature in a total volume of 1 ml using a 1cm i light path at 340 nm. Each cuvette contained: Enzyme, 75 mM TES buffer, 5 mM DTT,
+ 7.5 mM NAD , 10 mM malate and 1.5 mM MnCl0. Fig. (17) Purification of malic enzyme from Jerusalem artichoke mitochondria. Profile of malic enzyme activity ( ') eluted from Sephadex S-200 (A) and Sephadex A-25 (B) together with protein ( ). -94-
and showed activity with NAD and required Mg or Mn for activity.
It was also free of malate dehydrogenase. Requirement of malic enzyme
to bivalent cations was shown by many workers (Macrae 1971b; Davies and Patil 1975; Dittrich 1976).
+ Effect of NAD concentration
The plot of rate versus NAD+ concentration showed normal
Michaelis-Menton kinetics. The effect of increasing NAD+ concentrat- ion on the rate of malic enzyme activity is illustrated in Fig. (18).
Using two different concentrations of malate (10 and 50 mM, not chelated to metal), had the same effect on the response of malic en- zyme to varying concentrations of NAD+. Fig. (19) illustrates the response of malic enzyme to varying concentrations of NAD+ in the presence of 50 mM malate.
Fig. (18) and (19) show that the response of malic enzyme to + 2+ 2+ varying concentrations of NAD In the presence of Mg or Mn Is the same at the two concentrations of malate used.
The Michaelis constant (Km) for malic enzyme from Jerusalem arti- choke mitochondria obtained from Lineweaver-Burk plot of fitted lines
1/v versus 1/S for NAD+ is illsutrated in Fig. (20) in the presence of 4 mM unchelated MnCl2 and Fig. (21) in the presence of 8 mM un- chelated MgCl2.
The Km for NAD+ obtained are shown below
Substrate +MnCl2 +MgCl2
NAD+(mM) 0.66 0.75
The concentration of uncomplexed metal or malate were calculated by first calculating the concentration of Mn-malate and Mg-malate complexes. Fig. (18) Activity of mitochondrial malic enzyme as a function of NAD concentration in the 2+ 2+ presence of Mn (°—° ) or Mg (•—• ). Activity was measured at 340 nm in 75 mM TES buffer (pH 7.0) and 10 mM malate was used. ViO ON I
mM NAD
Fj-g- 09) Activity of malic enzyme as a function of NAD+ concentration in the presence of Mn2+ (o—o ) or ]yjg —„ Activity was measured at 340 nm in 75 mM TES buffer (pH 7.0) and 50 mM malate was used. -97-
Fig. (20)
Lineweaver-Burlc plot of reaction velocity as a function of NAD concentration in the presence of MnCl^. Assay mixture contained 75 mM TES (pH 7.0), 10 mM malate, 4 mM MnCl2> 5 mM DTT and NAD* (0-5 mM). -98-
1/S (mM NAD)
Fig. (21) Lineweaver-Burk plot of reaction velocity as a function of NAD+ concentration in the presence of MgCl^. Assay mixture contained 75 mM TES buffer (pH 7.0), 10 mM malate, 8 mM MgCU, 5 mM DTT and NAD+ (0-5 mM) . -99-
+ Macrae (1971b) reported K for NAD of malic enzyme from cauli- flower mitochondria of 0.5 mM. Coleman and Palmer (1972) showed K.
+ . for NAD for the enzyme isolated from Jerusalem artichoke mitochon- dria of 0.57 mM. Davies and Patil (1975) using cauliflower mitochon- 2+ dria reported K. of 0.22 mM in the presence of Mn and 0.51 in the 2+ presence of Mg . Hirai (1978) reported a value of 0.67 for the mito- chondrial enzyme from citrus fruit.
Results obtained in this study showed values of the same magni- tude as those obtained by Coleman and Palmer (1972) and Hirai (1978) 2+ 2+ in the presence of Mn and slightly higher in the presence of Mg b. Purification of NAD+-malate dehydrogenase Protein fractions obtained between 60-87% ammonium sulphate of sonicated mitochondrial supernatant of the same preparation used to purify malic enzyme, was used for the purification of malate dehy- drogenase.
Three peaks of protein were eluted from the sephacryl S-200 column. The first peak of protein (fractions 10-13) contained little malate dehydrogenase activity. While the second peak of protein
(fractions 14-26) coincided with very high activity of the enzyme.
The third peak of protein (fractions 27-31) was with very little ac- tivity. The fractions with high, activity were pooled together and concentrated over night at 4°C using amicon concentrator; this step resulted in the loss of about 70% of the total activity of the enzyme.
The elute from the sephacryl S-200 was found to be cloudy, so It was run through a sephadex G-25 column, and the enzyme activity was col- lected in 18.5 ml.
A flow sheet of the purification of malate dehydrogenase from
Jerusalem artichoke mitochondria and protein recoveries are shown in Total volume Total activity Total protein Specific activity Stage ml ymol NADH/min rag Unit/mg protein
Sonic supernatant 220.0 46233.78 4203 11 i 45% AS supernatant 216.0 40353.0 1836.0 22
60% AS supernatant 212.0 40738.0 1038.8 39
60-87% AS fraction 6.7 45546.6 887.8 40
After S-200 60.0 35691.3 432.96 82
Before G-25 7.5 9947.5 94.4 105
After G-25 18.5 9220.3 79.8 116
After A-50 151.0 5826.3 26.3 224
Final preparation 5.25 6752.4 20.7 326
Table (13) Purification of malate dehydrogenase from Jerusalem artichoke mitochondria.
Activity was measured by following change in absorbance at 34Q nm. Assay
medium consisted of: 75 mM TES, 0.2 mM NADH, 0.5 mM OAA and the enzyme in 1 ml. -101-
Table (13). The sonicated mitochondrial supernatant was used as a standard to calculate the relative purification and protein recoveries.
An increase of specific activity of 32.6 fold over the level of sonic supernatant obtained from mitochondria was achieved. The final pre- paration had a specific activity of 326.2 units per milligram protein.
i. Assaying malate dehydrogenase
For the determination of the K^ for NAD+, it was essential to assay the activity of malate dehydrogenase in the forward direction.
Malate dehydrogenase was assayed at pH 7.5 and pH 8.5. At pH 7.5 the response of enzyme to malate addition was linear at first, then the rate decreased gradually as time proceeded, due to the accumulation of oxaloacetate. When glutamate together with the enzyme glutamate- oxaloacetate transaminase was included in the reaction mixture, the inhibitory effect of oxaloacetate was decreased, but the rate did not become linear.
At pH 8.5, the response of enzyme to the addition of malate was faster, but the rate tended to slow down with time. By the addition of glutamate and GOT, at pH 8.5, a fast linear rate was obtained.
Lowering the H+ allowed the reaction to proceed to a greater extent.
Two types of buffers, TES (pH 8.5) and Tris-HCl (pH 8.5) were used, and the enzyme was found to give higher rate when Tris-HCl
(pH 8.5) buffer was used. Fig. (22) shows the relationship between the reaction rates and enzyme concentrations which was- linear.
ii. Effect of substrate concentration
The plot of reaction rate versus malate or NAD+ concentration showed normal Michaelis-Menton kinetics (Figs. 23 and 24). The effect of varying malate concentrations on the rate of malate dehydrogenase activity in the presence of 5 mM NAD+ is illustrated in Fig. (.23), and -1 02-
jul enzyme
Fig.(22) The rate of malate dehydrogenase activity as a function of enzyme concentration. Assay mixture contained: 50 mM Tris-HCl buffer (pH 8.5), 10 mM + malate and 5 mM NAD . 0 u> 1
Fig. (23) Malate dehydrogenase activity as a function of malate concentration. Assay.mixture contained 50 mM Tris-HCl
buffer (p+H 8.5), 10 mM glutamate, 2 yl COT (2 mg/ml), 5 mM NAD and malate (0-50 mM). -104-
the response of malate dehydrogenase activity to varying concentrations + of NAD m the presence of 20 mM malate is shown in Fig. (24). The
Michaelis constant (Km) for malate dehydrogenase obtained from Line- weaver-Burk plot of fitted lines 1/v versus 1/S for NAD+ and malate + are illustrated in Figs. (25 and 26). The determined for NAD and malate were found to be 0.45 mM and 2.4 mM respectively.
Siegal and England (1962) reported a Michaelis constant for malate of 0.25 mM at pH 8.4 for beef heart enzyme and 0.099 mM for + +
NAD . Yoshida (1965) showed a Km for malate and NAD for malate de- hydrogenase of B, subtil-is at 0.9 mM and 0.14 mM respectively in 0.1 M
Tris-HCl buffer at pH 8.8. Tyagi et al. (1977) using M. ~PKlei re- ported for NAD+ of 0.459 mM and for malate of 0.8 mM. Goonwardena and Wilson (1979b) using malate dehydrogenase isolated from turnip mitochondria.reported a for malate of 8.3 mM.
Malate dehydrogenase isolated and purified from Jerusalem arti-
+ choke mitochondria showed a Km for NAD of the same magnitude as that reported for the enzyme isolated from M. Fheli (Tyagi et al. 1977).
The Km for malate was higher than that reported for the enzyme iso- lated from mammalian tissue or bacteria, but lower than that reported for the enzyme isolated from turnip mitochondria (Goonwardena and
Wilson 1979b). £ 0 1
+ Fig. (24) Malate dehydrogenase activity as a function of NAD concentration. Assay mixture contained: 50 niH Tris- HCl buffer (pH 8.5), 10 mM glutamate, 2 yl GOT (2 mg/ ml), 20 mM malate and NAD+ (0-5 mM). 0 CTv 1
Fig. (25). Lineweaver-Burk plot of reaction velocity as a + function of NAD concentration. Assay mixture
contained: 50 mM Tris7HCl buffer (pH 8.5), 10 mM glutamate, 2 yl GOT (2 mg/ml), 20 mM Malate and NAD* (0-5 mM). -107-
Fig. (26)
Lineweaver-Burk plot of reaction velocity as a function of malate concentration. Assay mixture contained: 50 mM Tris-HCl buffer (pH 8.5), 10 mM glutamate, 2 yl GOT, 5 mM NAD and malate (0-50 mM). -108-
DISCUSSION
Mitochondria isolated from Jerusalem artichoke tubers, like other plant mitochondria, readily oxidize malate in the absence of any sys- tem added to remove the oxaloacetate. The oxidation of malate is possible because of the presence of an NAD+-linked malic enzyme in isolated plant mitochondria (Macrae and Moorhouse 1970, Macrae 1971a).
Malate oxidation via the malic enzyme gives rise to pyruvate as the end product and the reaction is not subject to inhibition as a result of equilibrium consideration or due to the accumulation of products of the reaction. While the reaction catalyzed by malate dehydrogenase -12
K is reversible and the reaction equilibrium ( eq = 2.3 x 10 ) favours the reverse reaction causing the formation of malate from oxaloacetate and the oxidation of NADH. Therefore, malate oxidation via the malate dehydrogenase reaction is only possible if the reaction products are removed. The removal of oxaloacetate can be achieved either by tran-
samination with glutamate in the presence of glutamate-oxaloacetate
transaminase (GOT), or by condensation with acetyl-CoA in the presence of citrate synthase. The remaining product, NADH is removed by oxi- dation via the electron transfer chain resulting in oxygen consumption.
When oxygen consumption accompanying malate oxidation was assayed
in the absence of a system to remove oxaloacetate, the rate of oxygen uptake in the presence of excess ADP soon declined, this was assumed
to be due to the accumulation of oxaloacetate (Fig. 3A), preventing
the malate dehydrogenase from playing its full role. This interpre-
tation was confirmed when glutamate together with glutamate-oxaloace-
tate transaminase were included in the reaction medium and an almost constant rate of malate oxidation was obtained. In this case the -109-
oxaloacetate formed was transaminated with glutamate to give aspartate and therefore, oxaloacetate was prevented from accumulating in the reaction medium.
Inclusion of thiamine pyrophosphate (TPP) in the reaction medium
stimulated the rate of oxidation and a constant rate of oxygen uptake, in the presence of malate as the substrate was observed (Fig. 2A).
Thiamine pyrophosphate is known to be an essential cofactor for pyru- vate dehydrogenase (Sanadi 1963). In this study it is assumed that
in the presence of thiamine pyrophosphate, pyruvate produced as a re-
sult of malic enzyme activity is decarboxylated to give acetyl-CoA which will condense with oxaloacetate to give citrate in the presence of citrate synthase, thus displacing the equilibrium of the reaction catalyzed by the malate dehydrogenase and therefore more malate would be oxidized.
It is therefore assumed from the data presented in this thesis
that isolated Jerusalem artichoke mitochondria are apparently defici-
ent in thiamine pyrophosphate and that exogenous coenzyme can gain
entry to the matrix space and increase the decarboxylation of pyru- vate to produce larger amounts of acetyl coenzyme A which has the
effect of integrating more efficiently the two pathways of malate
oxidation. Thiamine pyrophosphate is a very polar, water soluble molecule and the method by which it gains access to the matrix space
is puzzling and requires further study. The possibility that plant mitochondria have low complements of coenzymes and may have systems
to move them from one compartment to another opens up intriguing possibilities for control of cellular metabolism that merit serious
consideration in the future research. -110-
In the absence of added cofactors the addition of high concen- tration of ADP (200 yM) will stimulate the rate of oxygen uptake accompanying the oxidation of malate, however, the rate will soon de- cline to a slower rate. If ADP was added in smaller concentrations
(66 yM), such that a state 4 condition (as defined by Chance and
Williams 1956) intervenes between additions, the apparent decline in the shorter state 3 rates was less apparent, although the successive state 3 rates will be lower than the initial state 3 rate. This sug- gests that when malate oxidation was achieved continuously in state 3, oxaloacetate was produced faster than it could be removed, thus caus- ing an elevated concentration and inhibition of the overall reaction.
While, when the reaction was achieved through a series of state 3 to state 4 transitions, the oxaloacetate formed during the state 3 seemed
to be removed during state 4. During state 4 the activity of the re-
spiratory chain is supressed and consequently the internal NADH con- centration would increase as a result of malic enzyme activity which
is not subject to equilibrium control under the conditions of assay.
The increase in the NADH level must result in lowering the level of
oxaloacetate if the malate dehydrogenase is in equilibrium. It was
also suggested that ATP stimulated the rate of oxaloacetate removal
during the oxidation of malate (Hulme et al. 1967; Goonwardena and
Wilson 1979a). Hulme et al. (1967) suggested that the effect of ATP
on. the rate of oxaloacetate removal might be due to it acting as a
substrate in the reaction catalyzed by phosphoenol pyruvate carboxy-
kinase.
1 . Effect of inhibitors
a. Effect of piericidin A -111-
The oxidation of NAD+-linked substrates by plant mitochondria is not completely sensitive to piericidin A (or rotenone) (Wilson and
Hanson 1969; Ikuma and Bonner 1967; Brunton and Palmer 1972; Day and
Wiskich 1974a, b; Palmer and Arron 1967). The variability in response to rotenone (or piericidin A), an inhibitor of electron transfer through the non-haem iron centres associated with the internal NADH dehydrogenase complex (Ragan and Garland 1971; Ohnishi et al. 1972), the rotenone sensitive site is closely associated with coupling at the first coupling site, and the decrease in the ADP/0 ratios caused by rotenone (Day and Wiskich 1974a, b; Palmer and Arron 1976) were taken to indicate that the inhibitor insensitive dehydrogenase was not coupled to the first site of oxidative phosphorylation and that two internal NADH dehydrogenases were involved in the oxidation of
NAD -linked substrates in plant mitochondria (Palmer 1976).
When piericidin A was added to Jerusalem artichoke mitochondria oxidizingraalate i n the absence of an oxaloacetate-removing system, a
severe inhibition of oxygen uptake was observed (Fig. 6B). In this
case it was assumed that piericidin A and the accumulated oxaloacetate
are synergistic in causing a severe inhibition of the rate of malate oxidation. When thiamine pyrophosphate was included in the reaction mixture to activate the pyruvate dehydrogenase and lower the level of oxaloacetate, piericidin A caused less inhibition and a piericidin A
resistant rate was observed to develop (Fig. 6A). It was concluded
from these results that when oxaloacetate was allowed to accumulate,
thus oxidizing the pyridine nucleotide pool, only the piericidin A-
sensitive NADH dehydrogenase was in operation. Thus it would appear
that malate can only be oxidized by the rotenone resistant dehydro-
genase if the oxaloacetate is removed by either condensation with
acetyl-CoA or transamination to aspartate. -112-
b. Effect of n-butylmalonate
Malate oxidation can be inhibited by n-butylmalonate, which in- hibits the transport of malate into mitochondria (Robinson and
Chappell 1967; Phillips and Williams 1973; Wiskich 1975). Phillips and Williams (1973) reported that n-butylmalonate is a competitive inhibitor of malate dehydrogenase. Wiskich (1975) showed that n-buty- lmalonate inhibits malate oxidation by inhibiting the transport of malate through the mitochondrial inner membrane, while malate dehydro- genase was little affected by this compound. Results obtained in this study showed that n-butylmalonate inhibited the state 3 rate of malate oxidation, apparently by inhibiting the transport of malate, since
this compound was found to have no direct effect on malate dehydro- genase activity. At a concentration of 15 mM butylmalonate and in
the presence of 10 mM malate, the rate of oxygen uptake was inhibited • by 49% (Fig. 7), while the same concentration of n-butylmalonate had
almost no effect on malate dehydrogenase activity (Table 4).
When malate oxidation in the presence of n-butylmalonate was
inhibited by piericidin A, the piericidin A-resistant rate was found
to be inhibited by n-butylmalonate. This result suggests that m the
absence of added NAD the piericidin A resistant oxidation of malate
is sensitive to inhibition by butylmalonate and is therefore consis-
tent with the presence of two internal NADH dehydrogenases associated
with the oxidation of NAD+-linked substrates (Palmer and Arron 1976).
In contrast, the NAD+-stimulated rate of malate oxidation was
found to be unaffected by n-butylmalonate (Table 3). Day and Wiskich
(1974a) reported that malate oxidation both in the presence and ab-
sence of exogenous NAD* was equally sensitive to n-butylmalonate.
Suggesting that malate oxidation takes place in the matrix space and -113-
that reducing equivalents from internal NADH are transferred across the inner membrane by a transmembrane transhydrogenase and exogenous
NAD reduced on the outer face of the inner membrane. Goonwardena and •f Wilson (1979c) using turnip mitochondria reported that exogenous NAD increased the butylmalonate-insensitive component of malate oxidation and suggested that this component is transport-independent.
Results obtained in this study and illustrated in Fig. (8) and
Table (3) suggest that Jerusalem artichoke mitochondria oxidize mal- ate through two pathways; one is transport-dependent, i.e. takes place in the matrix space, and is associated with the internal NADH dehydro- genase system. While the other pathway is transport-independent re- quire the addition of NAD+ and is associated with the external NADH dehydrogenase system. Rustin et al. (1980) showed that the addition + . of NAD to plant mitochondria oxidizing malate in the presence or ab- sence of rotenone increased the rate of pyruvate production. Palmer and Arron (1976) also showed that in the presence of piericidin A, added NAD+ increased the rate of pyruvate production. Suggesting that added NAD+ stimulated malate oxidation via malic enzyme. In this study the stimulation by NAD+ was independent of malate transport
(butylmalonate-insensitive). This might indicate that added NAD+ was stimulating a component of malic enzyme which'seems to be located out- side the matrix compartment. c. Effect of oxaloacetate
Douce and Bonner (1972) reported that, when oxaloacetate was added to plant mitochondria oxidizing malate under the state 4 con- ditions it caused a strong, but transient inhibition of the rate of oxygen uptake. They suggested that the NADH produced by malic enzyme could be diverted from the electron transfer chain to reverse the -114-
malate dehydrogenase and when the oxaloacetate had been reduced, the
NADH once again became available to the respiratory chain and oxygen
uptake became evident. Brunton and Palmer (1973) showed that oxalo-
acetate added to wheat mitochondria respiring malate under state 3
conditions caused only a partial, but permanent, inhibition of the
rate of oxygen uptake. They suggested that the NADH produced from malic enzyme activity was not available for the reversal of malate
dehydrogenase to reduce oxaloacetate.
When oxaloacetate was added to Jerusalem artichoke mitochondria
oxidizing citrate it caused a powerful but transient inhibition (Fig.
9), and the rate of oxygen uptake was soon recovered to the initial
rate. It was concluded from this observation that the NADH produced
as a result of citrate oxidation was available for the reversal of
malate dehydrogenase activity and when oxaloacetate was reduced the + NAD pool became reduced and the rate of oxygen uptake recovered. On
the other hand when oxaloacetate was added to Jerusalem artichoke mito-
chondria oxidizing malate (Fig. 10B), it caused a partial inhibition,
but the rate of oxygen uptake never recovered to the initial rate.
This observation might suggest that part of the NADH produced as a re-
sult of malate oxidation was not available for the reduction of oxa-
loacetate and is not diverted from the respiratory chain.
When Jerusalem artichoke mitochondria oxidize malate the addition
of oxaloacetate will cause an equilibrium to be reached, i.e.
[Mai] [NAD+] * Since the level of malate is higher, [ OAA] [ NADH] can
be higher. When citrate is the substrate, the addition of oxaloa-
cetate will cause the malate dehydrogenase to go backward. Since no
malate is originally present, the [Mai] component of the equilibrium
would be much lower and thus the [ OAA ] [ NADH] will be correspondingly -115-
lower. If NADH is much lower the rate of respiration would be lower.
Thus different compartments proposed by Brunton and Palmer (1972) may
not be necessary.
+ 2. The effect of exogenous NAD
-f- . .... The addition of exogenous NAD to plant mitochondria oxidizing malate increased the rate of oxygen uptake. It was also noticed that + . . . added NAD relieved the piericidin A inhibition of malate oxidation.
The effect of NAD* on malate oxidation was reported by many workers
(Brunton and Palmer 1973; Palmer and Arron 1976; Day and Wiskich 1974
a, b; Neuberger and Douce 1978; Tobin et al. 1980). Neuberger and
Douce (1978) suggested that NAD* is transported through the inner mem-
brane and that the rate of transport is dependent on the initial con-
centration of NAD* in the matrix space. Neuberger and Douce (1978)
suggested that the addition of exogenous NAD* caused an increase in
the level of the coenzyme in the matrix and they further suggested
that the level of endogenous NAD* was too low to saturate the malic
enzyme and that entry of NAD* raised the coenzyme level sufficiently
to enable the malic enzyme to work at maximal velocity. Thus the in-
crease in the internal NAD* will activate the malic enzyme which
would produce more pyruvate and in the presence of pyruvate dehydro-
genase will give acetyl-CoA and relieves the inhibition caused by
oxaloacetate. Tobin et al. (1980) reported that mitochondria con-
taining low level of NAD*, such as potato mitochondria will respond
more to added NAD*, while mitochondria containing higher level of
NAD* such as mung bean mitochondria will respond less to added NAD*. + They suggested that NAD will enter the mitochondria and increase + the NAD /NADH ratio and thus displaces the equilibrium of malate -116-
dehydrogenase, which will produce more oxaloacetate.
Srere (1980) reported that actively respiring rat liver mito- chondria (state 3) have a matrix volume of approximately half that of non-respiring mitochondria (state 4). He suggested that this repre- sents the exit of almost all the free water in the matrix, resulting in a change in the concentration of metabolites and substrates. The apparent conentrations of soluble components in the matrix space may be very variable and thus the above explanation is difficult to justify.
Day and Wiskich (1974a, b) attributed the response of malate + . . oxidation to added NAD , to the stimulation of the activity of an in- ternal enzyme. They suggested that malate oxidation takes place in the matrix space and that reducing equivalents from endogenous NADH are transferred across the inner membrane by a transmembrane trans- hydrogenase, thus exogenously added NAD+ is reduced in the intermem- brane space and the resulting NADH is reoxidized by a specific NADH dehydrogenase located on the outer surface of the inner membrane.
This conclusion was based on the observation that malate oxidation both in the presence and absence of NAD+ was equally sensitive to n- butylmalonate and added inorganic phosphate. Results obtained in this study showed that, the state 3 rate of malate oxidation was sensitive to n-butylmalonate (i.e. transport dependent), while the NAD -stimul- ated rate was insensitive to inhibition by n-butylmalonate (transport- independent) . Palmer and Arron (1976) showed that inorganic phosphate was necessary for the oxidation of malate in the absence of added
NAD+ but was not necessary for the malate oxidation stimulation by exogenous NAD+. In addition phosphate is known to be an activator of mitochondrial malate dehydrogenase (Blonde et al. 1967). Day and -117-
Wiskich (1974a, b) reported that exogenous NAD+ relieved rotenone in- hibition of the rate of oxidation of citrate and a-ketoglutarate in the same manner as that of malate. In this study, results obtained + showed that the addition of NAD caused a stimulation of oxygen uptake when no substrate was added and the stimulation of citrate oxidation by added NAD+ in the presence of piericidin A was not greater than that of the control. On the other hand when malate was the substrate,
NAD+ stimulation was much higher than that of citrate and the control.
From these results, it was suggested that added NAD+, in the presence of piericidin A, was stimulating a component unique for malate oxi- dation, and is not common to all NAD+-linked substrates.
3. Reduction of exogenous NAD+
In intact plant mitochondria, malate is able to reduce exogen— ously added NAD+, while citrate fails to induce any reduction (Table
11). Day and Wiskich (1978) suggested that malate dehydrogenase is + responsible for the reduction of exogenous NAD . Malate oxidation takes place in the matrix and reducing equivalents are transferred across the membrane and the exogenous NAD+ is reduced in the inter- membrane compartment. They also showed that citrate failed to reduce exogenous NAD+. They attributed the failure of citrate to reduce exogenous NAD+ to the fact that in the presence of antimycin A, the
NADH produced as a result of citrate oxidation was not oxidised by the respiratory chain and that its accumulation would inhibit the citrate oxidation and as a result citrate would not produce high enough concentration of NADH to bring about the operation of the
transmembrane transhydrogenase.
Brunton and Palmer (1972) showed that wheat mitochondria oxidize malate and citrate and both substrates were able to reduce equal -1 18-
+ amounts of endogenous NAD . If a transmembrane transhydrogenase was + + involved in the reduction of exogenous NAD , all NAD -linked substrates would be expected to reduce exogenously supplied NAD*. + Results obtained in this study (Table 11), suggest that NAD re- duction by intact mitochondria seem to be induced by externally lo- cated enzymes. The biphasic manner of NAD* reduction by malate sug- gests that the initial fast rate is probably due to the activity of malate dehydrogenase. As in the washed preparation the initial rate was stimulated when a system for the removal of oxaloacetate was in- cluded in the reaction mixture, while the final steady rate was almost unaffected and it was assumed to be due to the activity of malic en- zyme.
The activity of malate dehydrogenase is probably due to a con- taminating soluble malate dehydrogenase released from broken mito- chondria. Although the preparation was highly intact, about 5% were apparently broken and Jerusalem artichoke mitochondria were found to contain a larger component of malate dehydrogenase than malic enzyme
(130:1) when both enzymes were isolated and purified from the same amount of tubers.
When mitochondria were purified through a Percoll density gradi- ent, the preparation was highly intact and broken mitochondria were reduced from 9% to 3%. Purified mitochondria reduced exogenously supplied NAD* in a biphasic maimer similar to that characteristic of washed mitochondria, however the initial rate of reduction was greatly reduced (Fig. 16 and Table 11), suggesting that the Percoll gradient had removed some of the soluble enzyme from the preparation. The de- crease in the initial rate of NAD* reduction was taken to indicate that the initial rate of reduction was due to the activity of soluble malate dehydrogenase released from broken mitochondria. Although, the -119-
percentage of broken mitochondria in the purified preparations was very low (3%) malate dehydrogenase is a very active enzyme and the re- lease of 3% of the matrix protein will result in a significant rate + . + of NAD reduction in the external phase if NAD was added. In con- trast, the final steady rate of NAD+ reduction was slightly increased (Fig. 16 and Table 11) upon purification and this rate was assumed to be due to the activity of malic enzyme possibly present in the inter- membrane compartment (Coleman and Palmer 1972; Brunton and Palmer 1973). This suggestion is consistent with the observation of Goonwardena and Wilson (1979b) who found that malic enzyme activity was not lost during the preparation of submitochondrial particles from turnip mitochondria and they assumed that it is membrane-bound enzyme. When intact mitochondria were allowed to reduce exogenously + supplied NAD , then centrifuged, the NADH formed was found in the + supernatant. Suggesting that added NAD was reduced outside the mat- rix compartment.
4. Purification of mitochondria
Percoll which is a silica sol coated with a layer of polyvinyl- pyrrolidol (PVP) was shown to be a useful medium for the separation of cells and cell organelles (Pertroft et al. 1977; Pertroft et al.
1978). Pertroft et al. (1977) compared different gradient materials and showed that Percoll has the advantage of being of high density, having low osmolarity because of its very high molecular weight and non toxic to animal tissues. These advantages made it superior to other gradient media, they also showed that it can be easily removed from the organelles by washing with regular washing medium. Since the experiments described in this thesis were carried out, other -120-
workers have reported the use of Percoll to purify mitochondria from
different plant tissues (Jackson et al. 1979; Bergman et al. 1980;
Goldstein et al. 1980). Jackson et al. (1979) purified mitochondria
from different plant sources and their mitochondrial preparations
contained very low chlorophyll contaminents. Bergman et al. (1980)
using a combination of phase-partition and Percoll density gradient
could obtain mitochondrial preparations free of chlorophyll.
During the course of this study, purification of mitochondria
from different plant sources by the utilization of Percoll density
gradient yielded a preparation of highly intact organelles. Puri-
fication of Jerusalem artichoke mitochondria on a 20% Percoll gradient
yielded two bands of mitochondria. Mitochondria of both bands were
of good quality according to intactness, ADP/0 and respiratory control
ratios. Goldstein (1980) using mitochondria isolated from wheat and
purified using a linear Percoll density gradient obtained mitochondria
in two bands of physiologically distinct activity. As the main object
of this study was to obtain purified intact mitochondria free of
soluble enzymes, lower concentrations of Percoll (18%) was used to
recover mitochondria in a single band. On the other hand mitochondria
isolated from potato tubers and mung bean hypocotyls formed a single
band on a 20% Percoll density gradient. Mitochondria purified from
different plant sources showed ADP/0 and respiratory control ratios
higher than those obtained with washed mitochondria.
Mitochondria isolated from Jerusalem artichoke and mung bean
hypocotyls purified on Percoll density gradient showed an increase in
the rate of oxygen uptake when NADH was supplied as a substrate indi-
cating the removal of non-mitochondrial protein. When malate was
supplied as a substrate purified mung bean mitochondria showed an -121-
increase in the rate of oxygen uptake, while Jerusalem artichoke mito- chondria showed no change in the rate of oxygen uptake. Upon purifi- cation mung bean mitochondria showed an increase in the concentration
+ of endogenous NAD of two fold while purified Jerusalem artichoke mitochondria showed an increase of 50%. This might indicate that washed preparation of mung bean mitochondria were more contaminated with soluble protein than Jerusalem artichoke mitochondria.
5. Malate oxidizing enzymes
Activity of NAD+-linked malic enzyme has been detected in cauli- flower mitochondria (Macrae 1971a, b), in some C^ and C^ plants uti- lizing aspartate for a source of CO^ (Hatch et al. 1975). Dittrich
(1976), carried out a survey of plants with crassulacean acid meta- bolism and showed that NAD-linked malic enzyme was present in plants investigated. Purification of mitochondria using sucrose density + gradients showed that NAD -malic enzyme was localized in mitochondria.
Malic enzyme isolated from cauliflower bud mitochondria showed activi- 2+ 2+ + ty with both Mn and Mg (Macrae 1971b), while NAD -malic enzyme 2+ from C^ plants showed an absolute requirement for Mn which could not 2+ be replaced by Mg (Hatch et al. 1974). Thus suggesting that there is a subtle difference between the malic enzymes present in these tissues.
Jerusalem artichoke mitochondria were shown to contain both NAD*- linked malic enzyme and malate dehydrogenase (Coleman and Palmer .4. 1972). The NAD -malic enzyme isolated and purified from Jerusalem artichoke mitochondria, by the use of Sephacryl S-200 and Sephadex 2+ 2+ A-25 showed requirement for Mg or Mn as a cofactor. The rate of malic enzyme activity as a function of varying concentrations of NAD* -122-
+ showed a normal Michaelis-Menten kinetics. The apparent K for NAD m of malic enzyme from Jerusalem artichoke mitochondria was 0.66 mM in 2+ . 2+ the presence of Mn and 0.75 mM in the presence of Mg . These values are comparable to those obtained by Coleman and Palmer (1972) and
Hirai (1978) for the enzyme isolated from citrus fruit. + ... NAD -linked malate dehydrogenase isolated and purified from
Jerusalem artichoke mitochondria showed a normal Michaelis-Mentcn kinetics when assayed in the presence of varying concentrations of
NAD+ or malate. When assayed at pH 8.5, NAD+ reduction was linear at first, but tend to slow down with time, this was attributed to the accumulation of oxaloacetate. When assayed in the presence of gluta- mate and glutamate-oxaloacetate transaminase, the rate was linear.
The rate of reaction showed a linear relation in the presence of dif- ferent concentrations of the enzyme.
Malate dehydrogenase showed an apparent K for malate of 2.4 mM, a value similar to that reported by Bowman and Ikuma (1976) for the enzyme isolated from mung bean mitochondria, but lower than that re- ported for the enzyme from turnip mitochondria (Goonwardena and Wilson
1979b) and higher'than the value reported for enzyme from mammalian tissue (Siegal and England 1962) or bacteria (Yoshida 1965). The Km + for NAD determined for malate dehydrogenase was 0.45 mM which was lower than that reported by Bowman and Ikuma (1976), who reported a value of 1 mM, but similar to that reported by Tyagi et al. (1977).
It has generally been agreed that plant mitochondria oxidize malate in the absence of a system for the removal of oxaloacetate produced as a result of malate dehydrogenase activity. This is due to the fact that plant mitochondria oxidize malate through two path- ways. One via malate dehydrogenase and the other via NAD+-linked -1 23-
malic enzyme, giving oxaloacetate and pyruvate respectively. Pyru- vate in the presence of pyruvate dehydrogenase would give acetyl-CoA which condenses with oxaloacetate giving citrate, thus displacing the
equilibrium towards malate oxidation. The combination of these two
enzymes together with pyruvate dehydrogenase and citrate synthase made it possible for the efficient oxidation of malate by plant mito-
chondria.
It is considered that malate dehydrogenase is controlled by its
equilibrium characteristics and that it is the malic enzyme which has
the complex regulatory properties (Wedding et al. 1976; Bowman and
Ikuma 1976). It was shown that the malic enzyme from mung bean
mitochondria is inhibited by NADH and activated by coenzyme A and sul
phate (Macrae 1971b). This was interpreted as to indicate a control
mechanism. The ability of plant mitochondria to produce pyruvate
from malate provides means by which plant mitochondria can oxidize
Krebs-cycle intermediates without the necessity of supplying pyru-
vate from glycolysis (Palmer 1976), specially under conditions, where
quantities of malate or citrate may be stored (Hirai 1978).
Results obtained in this study showed that Jerusalem artichoke
mitochondria oxidize malate through two pathways, one is transport-
dependent (butylmalonate-sensitive) and the other is transport-in-
dependent (butylmalonate-insensitive) and which requires the addition
+
of exogenous NAD . These observations together with the ability of
intact Jerusalem artichoke mitochondria to reduce exogenously
supplied NAD+ in the presence of malate, but not in the presence of
citrate, are consistent with the external location of a component of
enzymes responsible for malate oxidation and could not be explained
on the basis of transmembrane transhydrogenase activity. Recently -124-
Rustinetal. (1980) suggested that some malate dehydrogenase activity was found outside the matrix compartment. In this study the activity of malate dehydrogenase appeared to be due to a contaminating soluble malate dehydrogenase, as this activity was reduced when mitochondria were purified on Percoll density gradient. + The K^for NAD of malic enzyme and the malate dehydrogenase purified from Jerusalem artichoke mitochondria were almost of the same magnitude and the malic enzyme does not seem to require higher con- centration of NAD* as suggested by Neuberger and Douce (1978). Also the endogenous NAD* contents was about twice the Km value for malic enzyme.
At least with malate, the substrate used in this study, the evi- dence favour the presence of external component of malate oxidizing system, which might be a malic enzyme. -1 25-
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THE INHIBITION OF MALATE OXIDATION BY OXALOACETATE IN JERUSALEM ARTICHOKE MITOCHONDRIA
JOHN M. PALMER, RICHARD C. COWLEY and NAJAT A. AL-SANfS Department of Botany, Imperial College, Prince Consort Road, London SW7 2BB, England
ABSTRACT Some of the characteristics of malate oxidation by Jerusalem artichoke mito- chondria have been studied. At pH 6.5 the activity of malate oxidase in the presence of ADP was constant while at pH 8.0 it decreased. The addition of oxaloacetate to mitochondria at pH 8.0 under state 3 conditions caused a power- ful and transient inhibition of citrate oxidase and a partial but permanent in- hibition of malate oxidase. Under state 3 conditions the level of exogenous oxaloacetate rapidly decreased in the presence of citrate and increased in the presence of malate. When malate was oxidized in the presence of piericidin A the addition of oxaloacetate caused an inhibition of oxygen consumption similar to that observed with citrate in the absence of piericidin A. The data are in- terpreted as indicating that the endogenous NAD+ does not act as a single homo- genous pool.
INTRODUCTION The ability of plant mitochondria to oxidize malate in the absence of a sys- 1-3 tem to remove oxaloacetate has been observed by many workers and is a major difference between plant and animal mitochondria. The ability of plant mito- chondria to oxidize malate is considered to be due to either their ability to remove the oxaloacetate produced or to oxidize malate by an enzymic system other 4 5 than the malate dehydrogenase. Walker and Beevers and Avron and Biale pro- posed a system for the removal of oxaloacetate which involved the decarboxylat- ion of oxaloacetate to pyruvate and subsequent conversion to acetyl-CoA and NADH which could then be used to remove two further moles of oxaloacetate. Avron 5 and Biale demonstrated that in avocado mitochondria, in the presence of ADP, the NADH produced by pyruvate oxidation could be used to reduce oxaloacetate while NADH produced by the oxidation of malate continued to be oxidized by the respiratory chain. Wiskich and Bonner^observed that the activity of malate oxidase decreased in the presence of ADP, which they attributed to the accumulation of oxaloacetate. 118
Wiskich et at. and Lance et al. ' studied malate oxidation in avocado mito- 9 chondria and showed that under state 3 conditions (Chance and Williams ) the rate of oxygen uptake decreased while under state 4 conditions the rate increas- ed. They concluded that oxaloacetate accumulated during state 3 and disappear- ed during state 4 metabolism. The apparent accumulation of oxaloacetate could be prevented by adding thiamine pyrophosphate or glutamate to the assay medium Hulme et al.^ studied the metabolism of oxaloacetate in apple mitochon- dria and their data suggested that oxaloacetate was decarboxylated directly to pyruvate and that the NADH and acetyl-CoA produced from the metabolism of the pyruvate were used to reduce the concentration of oxaloacetate, as proposed by 4 5 10 Walker and Beevers and Avron and Biale . Hulme et al. also observed that oxaloacetate caused a transient inhibition of the rate of oxygen uptake result- ing from the oxidation of both malate and succinate and reported that oxaloace- tate was removed more rapidly in the presence of malate than succinate. 11 The discovery by Macrae and Moorhouse that plant mitochondria contained an NAD+-linked malic enzyme provided an explanation for the ability of malate to remove oxaloacetate. The malic enzyme is less sensitive to the accumulation of oxaloacetate and could provide NADH for the reversal of the reaction catalyzed by malate dehydrogenase. Data to support this concept were provided by Douce 3 and Bonner who showed that the transient inhibition of the oxygen uptake accom- panying the oxidation of malate and citrate, caused by adding oxaloacetate, was accompanied by the complete oxidation of the pyridine nucleotide pool. The NAD+ became reduced when the oxaloacetate had been removed and the oxidation rate recovered. These observations were made under state 4 conditions; the authors report that oxaloacetate will cause an inhibition of malate oxidase ac- tivity under state 3 conditions, however they do not make it clear whether the recovery from oxaloacetate inhibition occurs under state 3 conditions. Douce 3 and Bonner conclude that NADH produced by the malic enzyme is diverted from the respiratory chain to reduce oxaloacetate. 12 Brunton and Palmer , using mitochondria from wheat shoots, observed that under state 3 conditions oxaloacetate caused a powerful but transient inhibition of the rate of oxygen consumption when pyruvate or citrate were supplied as sub- strates and a partial but permanent inhibition when malate was the substrate. These data were interpreted as showing that, under state 3 conditions, the NADH produced by the malic enzyme was not freely available for reduction of oxaloace- tate. It therefore seemed that, under state 3 conditions, the NADH produced by the malic enzyme was not diverted from the respiratory chain to the malate dehy- drogenase when oxaloacetate was added. This evidence is similar to that ob- 119
5 12 tained by Avron and Biale . Brunton and Palmer also showed that if pierici- din A was added to mitochondria oxidizing malate a piericidin-resistant oxidat- ion developed and the NADH could be diverted completely from the respiratory chain to the malate dehydrogenase when oxaloacetate was added. The data presented in this paper show that data similar to those obtained by 12 Brunton and Palmer using wheat shoot mitochondria can be obtained using Jeru- salem artichoke mitochondria.
MATERIALS AND METHODS Jerusalem artichoke (Helianthus tuberosus) tubers were grown by the Botanical Supply Unit of London University. The tubers were cleaned and stored in sealed plastic bags at 4°C until needed. Mitochondria were isolated and oxygen con- 13 sumption measured as described by Palmer and Arron . In all assays 2.0 mg of protein were used in 1.0 ml. The levels of oxaloacetate were measured using the method of Williamson and 14 Corkey except that the fluorimetric assay was replaced by a spectrophotometric assay carried out using an Aminco DW-2 spectrophotometer at 340 nm. Protein estimation was carried out using the method of Lowry et al.^ after solubiliza- tion with 0.8% (w/v) deoxycholate.
RESULTS There is considerable evidence from data in the literature that the state 3 rate of oxygen consumption supported by malate oxidation decreases as the oxida- tion proceeds^. Figure 1 shows the response of malate oxidase at pH 6.5 and 8.0 to two additions of ADP. It is clear that at pH 6.5 there is no decrease in the velocity of the second state 3 rate which is taken to indicate no accumu- lation of oxaloacetate; at pH 8.0 the activity of malate oxidase after the second addition of ADP was substantially lower than the initial activity. This 16 result is in agreement with the observations of Macrae and the decreasing rate at pH 8.0 may be the result of a decreased malic enzyme activity at this pH. There is, however, reason to question whether an enzyme located in the matrix would show the same pH profile as the isolated enzyme. These data demonstrate the importance of pH when studying the oxidation of malate. Data in figure 2 show that under state 3 conditions at pH 8.0 the addition of oxaloacetate resulted in a powerful but transient inhibition of citrate oxidase activity and a partial but increasing inhibition of malate oxidase activity. Analysis of the oxaloacetate levels (figure 3) showed a removal of oxaloacetate in the presence of citrate and an accumulation of oxaloacetate in the presence 120
Fig. 1. The influence of pH on the state 3 activity of malate oxidase. The pH of the assay was 6.5 for trace A and 8.0 for trace B. The malate concentra- tion was 60 mM. State 3 was initiated by adding 250 pM ADP.
Time (seconds)
Fig. 2. The influence of oxaloacetate Fig. 3. Levels of oxaloacetate in on state 3 malate and citrate oxidases. the presence of malate (O) and cit- Initial concentrations of additions are rate (•). Conditions were as for malate 60 mM, citrate 20 mM, ADP 500 pM, fig. 3. Zero time was taken from oxaloacetate (OAA) 0.15 mM, at pH 8.0. when oxaloacetate was added. of malate. Thus the NADH produced by isocitrate oxidation appeared available for the reversal of the malate dehydrogenase while at least a proportion of the NADH produced by malate was not and remained available to the respiratory chain. These data strongly suggest some form of compartmentation of the NAD+ within the 12 matrix compartment. Brunton and Palmer proposed that the malate dehydrogen- ase and malic enzyme may exist in separate compartments in wheat shoot mitochon- dria. The effect of oxaloacetate on the rates of oxygen consumption due to malate oxidation at pH 6.5 and 8.0 and in metabolic states 3 and 4 is presented in fig- ure 4. It is clear that under state 3 conditions oxaloacetate only caused a partial inhibition of oxygen uptake which showed a slight tendency to recover at pH 6.5 but not at pH 8.0. Under metabolic state 4 conditions oxaloacetate 121
still caused only partial inhibition, however at pH 6.5 the inhibition was transient and the full rate soon returned, while at pH 8.0 there appeared to be no tendency to recover over the time scale employed. Thus under all conditions oxaloacetate only caused a partial inhibition of malate oxidase activity and complete reversal was observed only under state 4 conditions at pH 6.5.
Fig. 4. The influence of oxaloacetate on malate oxidase at pH 6.5 and 8.0. Oxaloacetate (0.15 mM) was added to traces (a) & (c) under state 3 conditions after adding 500 pM ADP and to traces (b) and (d) in state 4 conditions after the depletion of 150 p.M ADP. Malate concentration was 60 mM.
Fig. 5. The influence of piericidin A on malate oxidase in the presence of (a) no addition, (b) 0.75 mM thiamine pyrophosphate, (c) 10 mM glutamate and 2u glutamate-oxaloacetate transaminase, (d) thiamine pyrophosphate + glutamate + transaminase. Other additions were 60 mM malate, 2 mM ADP and 20 ng pieri- cidin A (P/A) per mg protein. 122
Previous studies^'^ have shown that at least two NADH dehydrogenases may be responsible for oxidizing the NADH produced from malate. Data in figure 5 show the complex response obtained when piericidin A was added to mitochondria oxid- izing malate under state 3 conditions. Malate can be oxidized by oxygen by two pathways, one sensitive and one resistant to inhibition by piericidin A. In the presence of glutamate and the transaminase piericidin A caused an immediate but partial inhibition of oxygen uptake. If no steps were taken to remove accumulated oxaloacetate then piericidin A caused a very powerful inhibition. However, after a period of time the resistant rate developed. The inclusion of thiamine pyrophosphate reduced the duration of the lag before the onset of the resistant rate and markedly increased the magnitude of the subsequent resistant rate of oxygen consumption.
The complete interpretation of these results is not yet possible but the lag which occurs between adding piericidin A and the onset of the resistant rate of oxygen consumption does appear to be a function of the level of accumulated oxaloacetate. It seems possible that in the presence of piericidin A reducing equivalents resulting from malate oxidation can be used to reduce oxaloacetate to malate. The data presented in figure 6 are consistent with this suggestion. From these two traces it can be seen that if oxaloacetate was added after pieri- cidin A it caused a very powerful inhibition of oxygen uptake which very quickly disappeared and the original rate returned. This suggests that in the presence of piericidin A all of the NADH produced from malate can be used to convert oxaloacetate to malate.
ADP 'ADP
Fig. 6. The influence of oxaloacetate and piericidin A on the activity of malate oxidase at pH 6.5. Additions were malate 60 mM, ADP 250 pM, oxaloace- tate 0.15 mM and piericidin A 20 ng/mg protein. 123
DISCUSSION
The most significant conclusion that can be drawn from the data presented is that the endogenous NAD+ in the mitochondria does not act as if it was in a single homogenous pool. Most of the NAD+ reduced by citrate can be diverted from the respiratory chain to reduce oxaloacetate, whilst under state 3 condi- tions a proportion of the NAD+ reduced by malate remained available to the res- piratory chain in the presence of oxaloacetate. In this respect Jerusalem 12 artichoke mitochondria appear similar to wheat shoot mitochondria . The data clearly show that under state 3 conditions oxaloacetate accumulated in the presence of malate while under state 4 conditions or in the presence of pierici- din A oxaloacetate competed effectively for the NADH produced from malate. The reason why oxaloacetate is a much more effective inhibitor of malate oxidation in the presence of piericidin A is unknown although Brunton and Palmer have 12 suggested one possible mechanism The data presented in this paper do not conflict with results already pub- lished. Avron and Biale^ showed oxaloacetate would not inhibit malate oxidat- 7 18 17 ion under state 3 conditions while Wiskich et al. , Lance et at. ' and Hobson report that oxaloacetate accumulated during state 3 respiration and disappeared during state 4 respiration. Both Hulme et al. ^ and Douce and Bonner"^ studied the influence of oxaloacetate under state 4 conditions and reported that it caused a transient inhibition of malate oxidase activity, in agreement with data 10 presented in figure 4 of this study. The data of Hulme et al. were also com- plicated because they added yeast concentrate, which contained NAD+ and thiamine pyrophosphate. The only real problem would appear to be the high degree of inhibition of malate oxidase activity caused' by oxaloacetate reported by Douce and Bonner; compare figure 4"^ with figure 4 in this paper. Unfortunately there are observations already published in the literature and presented in this paper which cannot be adequately explained. Of particular interest is the mechanism which allows the accumulation of oxaloacetate when malate is oxidized in the presence of 0.2 mM oxaloacetate (fig. 3). If citrate had been the substrate the reaction catalyzed by malate dehydrogenase would have run in reverse; even in the presence of 60 mM malate and piericidin A this reac- tion will run in reverse in the presence of oxaloacetate. Therefore the mech- anism by which oxaloacetate is accumulated under state 3 conditions is puzzling. Could it be that oxaloacetate is produced by a reaction other than that catalyz- ed by malate dehydrogenase? This possibility would explain how the concentra- tion of oxaloacetate rises to such a level that the malate dehydrogenase appears to run in reverse when ADP becomes exhausted or when piericidin A is added. 124
The possibility of compartmentation of the NAD+ involved with malate oxidat- ion catalyzed by either the malic enzyme or the malate dehydrogenase has defin- ite attractions when considering the way in which the balance of malate oxidat- ion by these two enzymes is regulated.
ACKNOWLEDGEMENTS This research was supported by grants from the Science Research Council, The Royal Society and the Central Research Fund, University of London. We would like to thank Mrs Jill Farmer for expert technical assistance and for prepar- ing the manuscript.
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